Am J Physiol Gastrointest Liver Physiol 317: G793–G801, 2019.

First published September 23, 2019; doi:10.1152/ajpgi.00199.2019.

RESEARCH ARTICLE Neurogastroenterology and Motility

Enteric neuron density correlates with clinical features of severe gut

dysmotility
Elisa Boschetti,1 Carolina Malagelada,2 Anna Accarino,2 Juan R. Malagelada,2 Rosanna F. Cogliandro,1
Alessandra Gori,1 Elena Bonora,1 Fiorella Giancola,1 Francesca Bianco,1 Vitaliano Tugnoli,3
Paolo Clavenzani,4 X Fernando Azpiroz,2 Vincenzo Stanghellini,1 Catia Sternini,5 and Roberto De Giorgio6
1
Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; 2Digestive System Research Unit,
University Hospital Vall d’Hebron, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
(Ciberehd), Barcelona, Spain; 3Department of Biomedical and Neuro Motor Sciences, University of Bologna, Bologna, Italy;
4
Department of Veterinary Medicine, University of Bologna, Ozzano, Italy; 5Digestive Disease Division, Departments of
Medicine and Neurobiology, University of California, Los Angeles, California; and 6Department of Medical Sciences,
University of Ferrara, Ferrara, Italy
Submitted 12 July 2019; accepted in final form 19 September 2019

Boschetti E, Malagelada C, Accarino A, Malagelada JR, Cogli- that patients with severe gut dysmotility exhibited an increased
andro RF, Gori A, Bonora E, Giancola F, Bianco F, Tugnoli V, interganglionic distance associated with a decreased number of my-
Clavenzani P, Azpiroz F, Stanghellini V, Sternini C, De Giorgio R. enteric and submucosal neurons, which correlated with symptoms and
Enteric neuron density correlates with clinical features of severe gut clinical manifestations of deranged intestinal motility.
dysmotility. Am J Physiol Gastrointest Liver Physiol 317: G793–
chronic intestinal pseudo-obstruction; enteric neuron cell count; in-
G801, 2019. First published September 23, 2019; doi:10.1152/ajpgi.
terganglionic distance; severe gut dysmotility
00199.2019.—Gastrointestinal (GI) symptoms can originate from
severe dysmotility due to enteric neuropathies. Current methods used
to demonstrate enteric neuropathies are based mainly on classic
qualitative histopathological/immunohistochemical evaluation. This
study was designed to identify an objective morphometric method for INTRODUCTION
paraffin-embedded tissue samples to quantify the interganglionic Severe gut dysmotility is a clinical condition with pro-
distance between neighboring myenteric ganglia immunoreactive for
neuron-specific enolase, as well as the number of myenteric and
nounced impairment of gut propulsion induced by alterations
submucosal neuronal cell bodies/ganglion in jejunal specimens of of enteric neurons and/or glial cells, interstitial cells of Cajal
patients with severe GI dysmotility. Jejunal full-thickness biopsies (ICC), and muscle cells in the gastrointestinal (GI) tract (8, 23).
were collected from 32 patients (22 females; 16 –77 yr) with well- Chronic intestinal pseudo-obstruction (CIPO), characterized by
characterized severe dysmotility and 8 controls (4 females; 47–73 yr). severe and recurrent subocclusive episodes with clinical and
A symptom questionnaire was filled before surgery. Mann-Whitney U radiological findings mimicking mechanical obstruction, is a
test, Kruskal-Wallis coupled with Dunn’s posttest and nonparametric particularly challenging and morbid form of severe dysmotility
linear regression tests were used for analyzing morphometric data and (7, 33). Patients with CIPO manifest severe symptoms (e.g.,
clinical correlations, respectively. Compared with controls, patients chronic nausea, vomiting, abdominal distension, and constipa-
with severe dysmotility exhibited a significant increase in myenteric tion/diarrhea) responsible for body weight loss, often requiring
interganglionic distance (P ⫽ 0.0005) along with a decrease in the intravenous nutritional support (9). In addition to CIPO, severe
number of myenteric (P ⬍ 0.00001) and submucosal (P ⬍ 0.0004)
dysmotility includes severe functional GI disorders, also re-
neurons. A 50% reduction in the number of submucosal and myenteric
neurons correlated with an increased interganglionic distance and
ferred to as enteric dysmotility; these patients present with
severity of dysmotility. Our study proposes a relatively simple tool severe motility impairment symptoms, nutritional defects, and
that can be applied for quantitative evaluation of paraffin sections impaired quality of life similar to CIPO but without intestinal
from patients with severe dysmotility. The finding of an increased subocclusive episodes (7, 22, 35).
interganglionic distance may aid diagnosis and limit the direct quan- A recent study by our group, comparing small bowel mano-
titative analysis of neurons per ganglion in patients with an intergan- metric abnormalities with histopathological findings in full-
glionic distance within the control range. thickness intestinal biopsies (24), showed that ~38% of the
NEW & NOTEWORTHY Enteric neuropathies are challenging patients with a neuropathic manometric pattern compatible
conditions characterized by a severe impairment of gut physiology, with severe dysmotility had apparently normal histopathology.
including motility. An accurate, unambiguous assessment of enteric However, our results did not establish a correlation between
neurons provided by quantitative analysis of routine paraffin sections intestinal manometry and histopathology. This unexpected
may help to define neuropathy-related gut dysmotility. We showed finding might be explained by the qualitative approaches tra-
ditionally used to demonstrate pathological changes through-
out the enteric neuromuscular layer in full-thickness biopsies,
Address for reprint requests and other correspondence: R. De Giorgio,
Dept. of Medical Sciences, Internal Medicine Unit, University of Ferrara,
which are not standardized and depend on an individual pa-
St. Anna Hospital, 1B1-1.35.16, Via A. Moro, 8-44124 Cona, Ferrara, Italy thologist’s expertise, often limited, with few exceptions. In-
(e-mail: [email protected]). deed, histopathology reporting and interpretation criteria are
http://www.ajpgi.org 0193-1857/19 Copyright © 2019 the American Physiological Society G793
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G794 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY

extremely subjective (3, 11). In clinically challenging condi- pain, nausea, vomiting, fullness, sensation of early satiety, and con-
tions, quantitative histopathological analysis may provide a stipation and/or diarrhea according to the Bristol stool scale. Extraint-
better definition of the underlying neuromuscular disease by estinal motor abnormalities, such as esophageal involvement and
revealing abnormalities that, although subtle, may bear diag- occurrence of gastroparesis, were also assessed. Finally, the presence
of small intestinal bacterial overgrowth (SIBO), urinary symptoms
nostic implications.
and age of symptom onset were evaluated by anamnesis and pertinent
The best strategy for a reliable quantitative analysis of diagnostic tests.
enteric neurons is to use whole mount preparations containing The study protocol was approved by the Ethics Committee of the
the myenteric and submucosal plexuses with neuronal cell University Hospital Vall d’Hebron, Barcelona, Spain, and the Ethics
bodies identified by panneuronal immunohistochemical mark- Committee of St. Orsola-Malpighi Hospital of Bologna, Italy (EM/
ers (4, 14, 17, 20, 26). However, this approach, which is often 146/2014/O). All patients gave written informed consent.
used in experimental animal models and in the research setting, Intestinal tissue sampling. Full-thickness biopsy specimens were
is not conveniently applicable in human routine pathology. obtained from the proximal jejunum either by laparoscopy or by
Indeed, virtually all surgical specimens are processed using conventional laparotomy. In each patient, a single circular segment (5
formalin fixation followed by paraffin inclusion (27). This cm long) was collected. Immediately after resection, specimens were
fixed in 10% formalin overnight, embedded in paraffin, and sectioned
routine method is reliable for pathological assessment, is con-
(5 ␮m) according to the standard protocol for tissue processing.
venient for long-term storage of the processed material, and Histopathology. A qualitative histopathological review was con-
entails limited costs. Furthermore, formalin-fixed, paraffin- ducted as previously described (24). Paraffin-embedded jejunal cross
embedded material can be sectioned by microtome and sec- sections were rehydrated, antigen retrieval was performed heating
tions can be often exchanged from one laboratory to another sections 25 min at 90°C in a water bath in the presence of 10 mmol/L
for referral, second evaluation and retrospective examination sodium citrate buffer pH 6.0 (Carlo Erba, Milan, Italy). Sections were
(10, 17). Thus, we considered such formalin-fixed, paraffin- treated with an endogenous peroxidase blocking kit (Gene Tex,
embedded specimens potentially suitable for neuron quantifi- Aachen, Germany). Immunostaining was performed using a commer-
cation. cial kit (Millipore, Milan, Italy) following the manufacturer’s instruc-
The enteric nervous system exerts a dominant regulatory tions.
role in the gut, making reliable quantitative enteric neuronal Histopathological material was examined blindly without prior
information about clinical and motility test outcomes. All biopsies
analysis a major target in any pathological evaluation of gut were first analyzed to exclude associated pathological findings such
specimens of patients with neuromuscular disorders. Nonethe- as malignancy, dysplasia, or villous atrophy. Routine histochemical
less, methodological criteria aimed at quantifying and assess- staining was used to evaluate the enteric neuromuscular architecture.
ing the density of enteric neurons are still largely debated, Immunohistochemical labeling was used to identify specific enteric
since an accurate evaluation of neuronal cell populations in neuromuscular component according to standardized and well-vali-
paraffin-embedded intestinal samples remains challenging. dated protocols (17). The following antibodies were used: 1) panneu-
This study was designed to establish a quantitative method ronal marker: neuron-specific enolase (NSE; rabbit PA1-28217, ready
using a panneuronal marker to analyze the number of neurons to use antibody; Thermo Fisher Scientific, Rockford, IL) and synap-
per ganglion in submucosal and myenteric plexuses along with tophysin (rabbit A0010, dilution 1:100; DAKO, Glostrup, Denmark);
the distance between myenteric adjacent ganglia in jejunal full 2) marker of cell survival: BCL-2 (mouse M0887, dilution 1:100,
DAKO); 3) marker of glial cells: S100␤ (rabbit Z0311, dilution 1:400,
thickness, formalin-fixed, paraffin-embedded biopsies from DAKO); 4) marker of ICC: c-Kit (rabbit A4502, dilution 1:400, DAKO);
patients with well-characterized severe gut dysmotility. We 5) marker of smooth muscle cells: ␣-smooth muscle actin (␣-SMA;
also tested for possible correlations between morphometric mouse M0851, dilution 1:400, DAKO); 6) markers of T lymphocyte
results and symptoms and clinical signs detectable in the subsets: CD45 (leukocytes; mouse M0834, dilution 1:40, DAKO),
enrolled patients. CD3 (general T lymphocyte marker; rabbit A0452, dilution 1:25,
DAKO), and CD8 (mouse M7103, dilution 1:50, DAKO; and 7)
MATERIALS AND METHODS marker of mast cells: anti-tryptase antibody (mouse M7052, dilution
1:800, DAKO).
Patients. Thirty-two patients with severe dysmotility (22 females; The immunohistochemical evaluation of neurons, glial cells, mus-
age range 16 –77 yr) were clinically evaluated at the Vall d’Hebron cle cells, ICC, and immune cells allowed us to stratify the patients.
University Hospital in Barcelona and recruited for this study between Three histopathological patterns were recognized in patients’ speci-
November 1999 and November 2009. Patients underwent abdominal mens according to the London classification (19):
surgery or laparoscopy, and full-thickness biopsies of the jejunum 1) apparently normal (AN): (n ⫽ 10; 7 females; age range: 24 – 61
were obtained. Patients with motility disorders secondary to infec- yr) no detectable changes in the neuromuscular layer;
tious, neurological, metabolic, autoimmune, and paraneoplastic con- 2) inflammatory neuromyopathy (INF): (n ⫽ 14; 10 females; age
ditions were excluded. Anorexia nervosa was ruled out in all patients range: 16 –77 yr) characterized by neuromuscular-glial-ICC al-
with malnutrition via psychiatric referral. The clinical diagnosis of terations with an inflammatory infiltrate throughout the neuro-
CIPO (n ⫽ 19; 12 females; age range 16 –77 yr) and enteric dysmo- muscular layer; and
tility (n ⫽ 13; 10 females; age range 25–52 yr) was established as 3) degenerative neuromyopathy (DEG): (n ⫽ 8; 5 female; age
previously described (24). Control patients (n ⫽ 8; 4 females; age range: 29 –70 yr) characterized by neuromuscular-glial-ICC al-
range 47–73 yr) underwent abdominal surgery for noncomplicated terations without any detectable inflammatory infiltrate through-
neoplastic tumor. Full-thickness biopsies were collected in the adja- out the neuromuscular layer.
cent jejunal tissue showing neither macroscopic nor microscopic The main findings for each of these patterns are detailed in a
abnormalities. Each patient and control subject answered a symptom previously published paper (24). A normal morphology was detected
questionnaire before surgery. The following data were collected: age, in jejunal samples from all control patients.
sex, body mass index (BMI), need for parenteral nutrition, central Interganglionic distance between myenteric ganglia. The distance
venous catheter-related infections, number of subocclusive episodes between myenteric ganglia was calculated on adjacent microscopic
(before surgery for each patient), abdominal distension, abdominal fields in random sections. All consecutive fields were analyzed from

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 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY G795
the first detectable ganglion present on one extremity of a given by three investigators (E. Bos, A. G., and R. De G., blinded in terms
section (beginning from the left side) toward the opposite site. At least of any clinical features or histopathological group from which the
three random (i.e., nonconsecutive) jejunal cross sections from one examined sections derived). Focusing on the neuromuscular ridge and
specimen per patient and per control were examined. Images (final moving along the myenteric ganglia as well as throughout the sub-
magnification ⫻1,000) were captured with a Nikon DXM1200 digital mucosal layer for related ganglia, we evaluated consecutive micro-
camera system (Nikon, Tokyo, Japan) on a LEICA DM LB (Leica, scopic fields (at least 20). Each blinded operator counted the total
Mannheim, Germany) light transmission microscope, coupled with number of NSE immunoreactive neuronal cell bodies/ganglion. Figure
the Automatic Camera Tamer (ACT)-1 dedicated software (Nikon). 2 shows representative examples of the sections and magnification
The distance between consecutive ganglia was calculated using the used for the neuronal count in the neuromuscular layer (Fig. 2A) and
software ImageJ v. 1.48. Because the actual margins of each ganglion in the submucosa (Fig. 2B).
in paraffin-embedded tissue sections cannot be firmly defined, we Interobserver variability for myenteric and submucosal neuronal
elected to consider clusters of NSE immunoreactive neuronal cell cell body count. To assess the interobserver variability of this tech-
bodies that were at a distance of less than 100, 200, 300, or 400 ␮m
nique, the final number of neuronal cell bodies/ganglion per patient
as a single arbitrary ganglionic unit. Each interganglionic distance
was calculated in myenteric and submucosal ganglia by each operator.
allowed us to identify significant differences in terms of neuronal
counts between pathological vs. control specimens. To establish a For myenteric ganglia, concordance among the data was defined when
reproducible criterion for neuronal counts, we chose the 300-␮m the difference in the mean number from each single count was no
distance, because, among the various ranges used in our paper, more than five neurons, whereas a difference of more than five
previous ancillary experiments led us to conclude that this was the neurons was defined as discordant. Because of the limited number of
closest interganglionic distance best fitting with the distance between neuronal cell bodies in submucosal ganglia, a mean number difference
ganglia evaluated by qualitative analysis (data not shown). The from each single count no more than one neuron was considered
quantitative data were correlated with symptoms and signs of patients concordant. To test the power of the method among observers, the
with severe dysmotility (Fig. 1). mean value of myenteric neurons per ganglion of samples that did not
Neuronal cell body count per myenteric and submucosal ganglion. reach concordance (n ⫽ 8) was included in the study. Since the mean
Myenteric and submucosal NSE immunoreactive neuronal cell bodies value of neurons calculated in patients with dysmotility was still very
were counted per ganglion at ⫻4,000 final magnification with a light far from the mean value of neurons in controls, the samples that did
transmission microscope (Olympus AX 70; Olympus, Melville, NY) not reach agreement were not outliers.

Fig. 1. Measurement of interganglionic distance. Schematic representation of the method by which interganglionic distances were defined, using the 300-␮m
cut-off value. Groups of immunoreactive neurons separated by less than 300 ␮m (distances indicated by *) were considered part of the same arbitrary ganglionic
unit. Distances ⱖ300 ␮m were used as the threshold value to distinguish two different ganglia (indicated by square brackets).

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G796 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY

Fig. 2. Neurons identified by a pan-neuronal marker in myenteric and submucosal plexuses. Representative photomicrographs showing neuron-specific enolase
(NSE) immunolabeling in the myenteric (A) and submucosal (B) plexus of a control sample. Black arrows indicate identified neuronal cell bodies.

Statistical analysis. The statistical analysis was performed using Wallis P ⫽ 0.0013) with no difference between the two
the free Software R-Cran (v. 3.5.2; Free Software Foundation’s GNU subgroups (Fig. 4, C and D). Quantitative analysis of enteric
project). To evaluate the differences between means (SD) of myen- neurons in patients with severe dysmotility subdivided into the
teric and submucosal neurons, as well as interganglionic distance, the
three histopathologically defined subsets (Fig. 4, E and F)
following tests were applied: 1) Mann-Whitney U test to compare
severe dismotility vs. controls; 2) Kruskal-Wallis coupled with a showed that neurons in myenteric plexus decreased 1.9-fold in
secondary Dunn’s comparative test for multiple group comparisons AN, 2.3-fold in INF, and 2.6-fold in DEG vs. controls (P ⫽
was used to assess the three main groups, i.e., CIPO vs. enteric 0.0106, P ⬍ 0.00001, and P ⬍ 0.00001, respectively; Kruskal-
dysmotility vs. controls and AN vs. INF vs. DEG vs. controls). Wallis P ⬍ 0.00001). Furthermore, INF and DEG showed
Univariate and multivariate nonparametric linear regression tests were fewer myenteric neurons than AN (P ⫽ 0.0390 and P ⫽
carried out to identify possible correlations between morphometric 0.008). The number of neurons/ganglion calculated in submu-
and clinical data. cosal plexus were decreased 1.7-fold in AN, 1.6-fold in INF,
and 1.4-fold in DEG vs. controls (P ⫽ 0.0001, P ⫽ 0.0003,
RESULTS P ⫽ 0.0496, respectively; Kruskal-Wallis P ⫽ 0.0006).
Interganglionic distance in myenteric ganglia. When we The final concordance of neuronal cell counts in myenteric
used 300 ␮m as the cut-off distance between two adjacent plexus of severe dysmotility and controls, obtained comparing
myenteric ganglia, sections of patients with severe dysmotility the final results of each operator, was 80% for the total 40 cases
(as a whole) exhibited a significantly increased interganglionic (patients and controls) analyzed in this study. The final con-
distance compared with controls (P ⫽ 0.0005; Fig. 3, A, D, and cordance of neuronal count in submucosal plexus was 97.5%.
F). The interganglionic distances in patients with enteric dys- Correlation among symptoms, neuronal counts, and inter-
motility or CIPO (Fig. 3B) were also significantly increased ganglionic distances. The number of subocclusive episodes
compared with controls (P ⫽ 0.0076 and P ⫽ 0.0001, respec- increased in INF and DEG vs. controls (P ⫽ 0.0025 and P ⫽
tively; Kruskal-Wallis P ⫽ 0.0009). There were no differences 0.0022; Kruskal-Wallis P ⬍ 0.01) but not in AN subgroup
in interganglionic distance between patients with enteric dys- (Fig. 5A).
motility or CIPO. Similarly, the interganglionic distance in Data analysis showed correlations between the number of
patients with with AN, INF, or DEG CIPO (Fig. 3C) was both myenteric and submucosal neurons per ganglion and
significantly increased compared with controls (P ⫽ 0.0099; various morphometric and clinical parameters.
P ⫽ 0.0004, and P ⫽ 0.0008, respectively; Kruskal-Wallis P ⫽ Univariate analysis indicated that the myenteric intergangli-
0.0035). The comparison of interganglionic distances among onic distance was inversely correlated with the number of
AN, INF, and DEG histopathological subgroups did not show myenteric neurons (t ⫽ ⫺2.953, P ⫽ 0.006); and directly
significant differences. correlated with the number of subocclusive episodes (t ⫽ 4.54,
Myenteric and submucosal neuronal counts. Compared with P ⬍ 0.001). Abdominal distension was inversely correlated
controls, patients with severe dysmotility showed a 2.2-fold with the number of neurons/ganglion in both myenteric and
decrease in the number of neurons per ganglion in myenteric submucosal plexuses and directly correlated with the intergan-
plexus (P ⬍ 0.00001) and a 1.6-fold decrease in submucosal glionic distance (t ⫽ ⫺6.066, P ⬍ 0.0001; t ⫽ ⫺3.768, P ⫽
plexus (P ⬍ 0.0004; Fig. 4, A and B). Similarly, in enteric 0.001; t ⫽ 2.268, P ⫽ 0.029). Symptoms showing an inverse
dysmotility and CIPO subgroups, neurons decreased 2.2-fold correlation with the number of neurons/ganglion in both my-
vs. controls in myenteric plexus (P ⬍ 0.00001 and P ⫽ 0.0001; enteric and submucosal plexuses included pain (t ⫽ ⫺4.947,
Kruskal-Wallis P ⬍ 0.00001) and 1.6-fold in submucosal P ⬍ 0.0001; t ⫽ ⫺03.692, P ⫽ 0.001); early satiety (t ⫽
plexus (P ⫽ 0.0014 and P ⫽ 0.0002, respectively; Kruskal- ⫺2.983, P ⫽ 0.005; t ⫽ ⫺2.16, P ⫽ 0.038), and constipation

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 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY G797

Fig. 3. Interganglionic distance in myenteric ganglia. Distance between ganglia using the 300-␮m threshold comparing controls (622.3 ⫾ 151.8 ␮m) and patients
with severe dysmotility (427.4 ⫾ 46.8 ␮m), *P ⫽ 0.0005 (A), enteric dysmotility (569.8 ⫾ 117.7 ␮m) and CIPO (653.3 ⫾ 164.7 ␮m), *P ⫽ 0.0076 and #P ⫽
0.0001 (B), and apparently normal (AN; 562.1 ⫾ 93.2 ␮m), inflammatory neuromyopathy (INF; 651.3 ⫾ 190.6 ␮m), and degenerative neuromyopathy (DEG;
645.25 ⫾ 134.2 ␮m), *P ⫽ 0.0099, #P ⫽ 0.0004, and §P ⫽ 0.0008 (C). Bottom: examples of interganglionic distance measurements using ImageJ software for
a control sample (D) and a patient with severe dysmotility (E). Neuron-specific enolase (NSE) immunostaining; 500 ␮m final magnification.

(t ⫽ ⫺3.215, P ⫽ 0.03; t ⫽ ⫺2.907, P ⫽ 0.006). Finally, SIBO suspected by altered small bowel or colonic manometry or by
and nausea correlated with the interganglionic distance (t ⫽ qualitative histopathological alterations with or without mor-
2.219, P ⫽ 0.033; t ⫽ 2.547, P ⫽ 0.015). phometric analysis of the number of enteric neurons. Although
Multivariate analysis of all covariates that had a significant whole mount preparations represent the gold standard for
value at univariate analysis, identified a correlation between submucosal and myenteric neuron quantitative analysis (12,
the interganglionic distance and the number of intestinal sub- 14, 26, 34), more readily available, routinely collected, paraf-
occlusive episodes (t ⫽ 2.742, P ⬍ 0.01; Fig. 5B) and an fin-embedded specimens may provide useful information on
inverse correlation between the number of neurons/ganglion in enteric neuron number. However, the validation of a standard
myenteric plexus and abdominal distension (t ⫽ ⫺2.542, P ⫽ method for quantitative analysis of enteric neurons on paraffin-
0.0167). embedded sections has long been debated among enteric neu-
ropathologists to better characterize patients with neurogenic
DISCUSSION
intestinal dysmotility (3). In this morphometric study, we
A complex integration among different regulatory cell types aimed to establish a method that would allow us to use
in the GI tract, including neurons and ICCs, is essential for the paraffin-embedded tissue samples for quantitative analysis tak-
coordination of smooth muscle activity, thereby tuning GI ing into account the number of neuronal cell bodies/(myenteric
motor function and propulsion (16, 30, 36). Various environ- and submucosal) ganglion as well as the distance between two
mental noxae and genetic factors may disrupt the delicate adjacent myenteric ganglia in jejunal specimens of patients
equilibrium underlying the physiology of gut function and with severe forms of gut dysmotility compared with controls.
cause dysmotility (3, 11, 18). Enteric neurons exert a dominant To overcome the difficulty of establishing the margin of each
role in the control of GI motility (16); thus, clinical conditions individual ganglion in bidimensional tissue preparations such
characterized by altered GI motility patterns are often referred as paraffin sections, we tested distances (from 100 to 400 ␮m)
to as enteric neuropathies (32). Enteric neuropathies can be between two clusters of myenteric neurons identified with a

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G798 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY

Fig. 4. Myenteric and submucosal neuronal cell counts in patients with severe dysmotility and controls. A: myenteric neurons counted in the whole severe
dysmotility (24.0 ⫾ 5.8 neurons/ganglion) group vs. controls (52.4 ⫾ 10.0 neurons/ganglions), *P ⬍ 0.00001. B: submucosal neurons counted in the whole
severe dysmotility (2.6 ⫾ 0.5 neurons/ganglion) group vs. controls (4.2 ⫾ 1.3 neurons/ganglion), *P ⫽ 0.0004. C: myenteric neuronal counts in enteric
dysmotility (ED; 23.8 ⫾ 5.5 neurons/ganglion) and chronic intestinal pseudo-obstruction (CIPO; 24.0 ⫾ 6.0 neurons/ganglion) subgroups, *P ⬍ 0.00001 and
#P ⬍ 0.0001. D: submucosal neuronal counts in ED (2.5 ⫾ 0.4 neurons/ganglion) and CIPO (2.7 ⫾ 0.5 neurons/ganglion) subgroups,*P ⫽ 0.0014 and #P ⫽
0.0002. E: myenteric neuronal counts in apparently normal (AN; 28.3 ⫾ 5.6 neurons/ganglion), inflammatory neuromyopathy (INF; 22.9 ⫾ 5.6 neurons/
ganglion), and degenerative neuromyopathy (DEG; 20.4 ⫾ 4.0 neurons/ganglion) subgroups, *P ⫽ 0.0106, #§P ⬍ 0.00001, $P ⫽ 0.0390, and °P ⫽ 0.008. F:
submucosal neuronal counts in AN (2.5 ⫾ 0.5 neurons/ganglion), INF (2.6 ⫾ 0.4 neurons/ganglion), and DEG (3.0 ⫾ 10.5 neurons/ganglion) subgroups, *P ⬍
0.0001, #P ⫽ 0.0003, and §P ⫽ 0.0496.

pan-neuronal marker within a ganglion to distinguish patients’ if further validated in a large cohort of samples, it will hope-
(disease) vs. controls’ (normal) specimens and elected to use a fully provide a reliable diagnostic tool for patients with severe
300-␮m interganglionic distance to quantify the number of neurogenic dysmotility.
myenteric neurons/ganglion. Notably, this quantitative method Except from guidelines by an international working group
showed a very low variability among different operators, and, on gut tissue collection, handling, and histopathological report-

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 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY G799

Fig. 5. Number of subocclusive episodes distribution among immunohistochemically defined subgroups and relationship with interganglionic distance. A: number
of subocclusive episodes recorded per patient before surgical intervention. Patients were clustered accordingly with the qualitative immunohistochemically
defined assessment. *P ⫽ 0.0025 and #P ⫽ 0.0022. B: scatter plot illustrating the relationship between number of subocclusive episodes and interganglionic
distances.

ing of patients with gut dysmotility (18), there are no indica- due to the lobulated silhouette of the human myenteric ganglia
tions as to how a neuronal quantitative analysis should be that often prevents an accurate definition of the ganglionic
performed on formalin-fixed, paraffin embedded specimens, perimeter. Pathologists have suggested to define the border of
the most commonly available material in any pathology unit. a ganglion when one or more neurons and glial cells are
The lack of a standardized quantitative method is the main surrounded by perineural cells (15). A shortfall of this crite-
reason behind the wide variability reported so far in studies rion, however, is the finding that neurons can also be found in
where neuronal counting has been attempted using tissue tertiary nerve fiber strands in the human colon (34). Other
specimens of patients with gut motility disorders (3). Boyer et definitions of a myenteric ganglion include the detection of two
al. (5) compared neuronal assessment in whole mounts vs. or more adjacent ganglion cell bodies (2) or the presence of a
paraffin-embedded tissue sections in a mouse model of dini- minimum of three neuronal cell bodies with an interperikarya
trobenzene sulfonic acid-induced colitis. The results, expressed distance not exceeding two cell body diameters (6). Our
as neuronal cell body/ganglion in paraffin-processed material, approach to overcome the difficulty of defining ganglion actual
and as neurons per square millimeter in whole mounts, showed, margins using paraffin-embedded material and microtome sec-
as expected, marked differences in absolute numbers due to the tions was to define arbitrary myenteric ganglionic units as
different methods, but the percentage of neuronal cell loss was clusters of neurons separated by a fixed distance that allowed
quite similar to that of the two quantitative approaches, thus us to identify significant differences in neuronal counts be-
demonstrating the validity of both methods for the determina- tween pathological and control specimens.
tion of neuropathological changes. A key accuracy factor when estimating the number of
One of the main issues in neuronal cell body number myenteric and submucosal neurons is the reproducibility in
evaluation in paraffin material is about data normalization. terms of interobserver-variability. In this study, the concor-
Many methods have been attempted, but a standard consensus dance among the three investigators was defined as a cut-off of
has not been achieved. Ippolito et al. (17) quantified myenteric up to five myenteric neuron cell bodies, i.e., about five times
neurons in paraffin section of human left colon in a control lower than the difference between controls and severe dysmo-
population, reporting the number of neurons as ⌺ neurons tility patients. The interobserver concordance was reached
identified by the pan-neuronal marker HuC/D/⌺ ganglionic when the final measurement of each operator did not exceed
area. Since myenteric ganglia are variable in size/area depend- five neuronal cell bodies from the average of three assess-
ing on the number of contained neurons or glial cells (13, 29, ments. Using this cut off we had only 20% discordance.
37), this quantitative method may yield misleading results Furthermore, among the group of discordant samples (8/40),
when one is comparing tissues from dysmotility patients to seven cases had six to ten neuronal cell bodies above the
controls. Mandic et al. (25) measured the number of myenteric concordant limit of five, whereas only one was discordant for
neurons per square centimeter of surface of the esophagus, more than ten neuronal cell bodies. However, the estimated
stomach, duodenum, jejunum, ileum, transverse colon, and discordance (20%) did not affect the final quantitative results
rectum in paraffin control tissue samples. However, a potential because of the stringent cut-off number (i.e., ⱕ5) of myenteric
limitation of this method, which measures neurons normalized neurons used to establish interobserver agreement.
per microscopic field, is neuronal overestimation because of In our study, we detected a marked loss of myenteric
the inclusion of extraganglionic myenteric perikarya, which neurons in all patients with severe dysmotility, which was
have been demonstrated to increase progressively with age (1). similar for the two clinically relevant phenotypes, CIPO and
Based on these data, enteric neuron counts fit best when enteric dysmotility, and for the three histopathological sub-
normalized per ganglion, although this does not overcome the types, i.e., AN, INF, and DEG. Our data, showing a substantive
problem of differences in ganglion size. However, this ap- reduction (~50%) of both myenteric and submucosal neurons,
proach implies a definition of ganglion (1, 29) that is difficult are in agreement with the few previously reported quantitative

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G800 QUANTITATIVE NEURONAL CHANGES IN ENTERIC NEUROPATHY

data in tissues of patients with severe dysmotility, including analysis of neuronal cell bodies per ganglion to patients with an
(28, 31) infants/children with CIPO (21). Although the sub- interganglionic distance within the range of controls. Patients
mucosal plexus does not play a central role in controlling who are labeled “apparently normal” by qualitative immuno-
gastrointestinal motility, the neuronal loss also affected the histochemistry may have underlying quantitative intergangli-
submucosal plexus. The decrease of neuronal cell bodies in- onic and neuronal number changes bearing clinically meaning-
volved both type of ganglia in the jejunum of patients with ful information.
severe dysmotility.
Another significant morphometric result in our study is that GRANTS
the decreased number of myenteric neurons correlated with the This study was supported by Grant GGP15171 from Fondazione Telethon
increase of interganglionic distance. Compared with controls, to E. Bonora and R. De Georgio and by University of Bologna and Ferrara
(Ricerca Fondamentale Orientata and Fondo per le Agevolazioni alla Ricerca
the distance between adjacent myenteric ganglia was increased funds, respectively) to R. De Georgio. R. De Georgio received research grants
by ~40% in severe dysmotility (either enteric dysmotility or from Fondazione Del Monte of Bologna and Ravenna. C. Malagelada received
CIPO) and in histochemically distinct severe dysmotility sub- research funds from the Spanish Ministry of Economy and Competitiveness
sets. From a clinical point of view, the distance between (Dirección General de Investigación Científica y Técnica, SAF 2016-76648-
ganglia may have considerable diagnostic potential since ~76% R). Ciberehd is funded by the Instituto de Salud Carlos III. P. Clavenzani
received research grants from Fondazione Cassa di Risparmio di Bologna. C.
of patients with severe dysmotility can be identified by inter- Sternini was supported by the Imaging and Stem Cell Biology Core, National
ganglionic distance and the remaining 24% by adding the Institute of Diabetes and Digestive and Kidney Diseases Grant P30 DK-41301.
quantitative analysis of neuronal number per ganglion. Future
studies based on a large control sample size, taking into DISCLAIMERS
account sex- and age-related factors, are needed to establish the The funding bodies did not influence the content of this article.
interganglionic distance range of control (normal manometric
pattern/absence of symptoms) subjects and validate objective DISCLOSURES
criteria for the diagnosis of enteric neuropathies. We believe R. De Georgio has participated as a consultant for Shire, Sucampo,
that an ideal quantitative approach should include the intergan- Coloplast, Kyowa Kirin International, and Takeda and received grant support
glionic distance as a first parameter, followed by neuronal cell from Shire and Takeda. These consultancies did not influence the content of
this article. The other authors declare no conflict of interest.
count in patients with normal interganglionic distance. Further-
more, in our study, the increased interganglionic distance AUTHOR CONTRIBUTIONS
correlated with a higher number of intestinal subocclusive E. Boschetti, V.S., C.S., and R.D.G. conceived and designed research; E.
episodes, thus implying that the distance between myenteric Boschetti, C.M., R.F.C., A.G., F.G., F.B., V.T., and P.C. performed experi-
ganglia might indicate the most severe forms of dysmotility. In ments; E. Boschetti, C.M., A.A., J.R.M., R.F.C., A.G., E. Bonora, F.G., F.B.,
addition, the altered GI motility and the worsening of gut V.T., P.C., F.A., C.S., and R.D.G. analyzed data; E. Boschetti, C.M., A.A.,
dysmotility, as reflected by an increased abdominal distension J.R.M., R.F.C., E. Bonora, F.A., V.S., C.S., and R.D.G. interpreted results of
experiments; E. Boschetti prepared figures; E. Boschetti, A.A., J.R.M., F.G.,
(7), were associated with at least 50% neuronal loss in the F.A., C.S., and R.D.G. drafted manuscript; E. Boschetti, C.M., A.A., J.R.M.,
small intestine. Overall, our data indicate that the clinical R.F.C., E. Bonora, F.A., V.S., C.S., and R.D.G. edited and revised manuscript;
manifestations and their severity increase as the myenteric E. Boschetti, C.M., A.A., J.R.M., R.F.C., A.G., E. Bonora, F.G., F.B., V.T.,
neuronal number decreases and the interganglionic distance P.C., F.A., V.S., C.S., and R.D.G. approved final version of manuscript.
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