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Developmental changes in submucosal nitrergic neurons in
the porcine distal colon
B
Sandra Montedonico, Thambipillai Sri Paran, Martina Pirker, Udo Rolle, Prem Puri*
Children’s Research Centre, Our Lady’s Hospital for Sick Children, Dublin 12, Ireland
Abstract
Background/Purpose:As our understanding of the enteric nervous system improves, it becomes clear
that it is no longer sufficient to simply determine whether enteric ganglion cells are present but also to
determine whether correct number and types of ganglion cells are present. Nitric oxide is recognized as a
potent mediator of inhibitory nerves responsible for the relaxation of the smooth muscle of the
gastrointestinal tract. The aim of this study was to determine the normal nitrergic neuronal density and
morphology in the submucosal plexus of the porcine distal bowel from fetal life to adulthood.
Methods:Distal large bowel specimens were obtained from porcine fetuses of gestational age E60 (n = 5),
E90 (n = 5), 1-day-old piglets (n = 5), 4-week-old piglets (n = 5), 12-week-old piglets (n = 5), and adult
pigs (n = 5). Whole-mount preparations of the submucosal plexus were made and stained with NADPH
diaphorase histochemistry. The ganglia density, the number of ganglion cells per ganglia, and nucleus
and cytoplasmic area were measured.
Results:Ganglia density decreased progressively and markedly with age until the adulthood ( Pb.001).
On the contrary, ganglion cells increased their size over time predominantly because of increase in
cytoplasm ( Pb.001). The number of ganglion cells per ganglia increased significantly during the fetal
life. However, there was a significant reduction in the number of ganglion cells per ganglia during the
period from birth to 4 weeks, remaining constant thereafter ( Pb.001).
Conclusions:The quantitative and qualitative morphometric analysis of the colonic submucous plexus
shows that significant developmental changes occur during fetal and postnatal life. These findings
indicate that the age of the patient is of utmost importance during histopathologic evaluation of enteric
nervous system disorders.
D2006 Elsevier Inc. All rights reserved.
Normal intestinal motility depends on the interaction of
the enteric nervous system and the smooth muscle cells in
the gut wall. As our understanding of the enteric nervous
system improves, it becomes clear that it is no longer
sufficient to simply determine whether enteric ganglion cells
are present or absent but also to determine whether the
correct number and type of ganglion cells are present at
different developmental stages.
0022-3468/$ – see front matter D2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jpedsurg.2005.12.063
Presented at the 37th Annual Meeting of the Canadian Association of
Paediatric Surgeons, Quebec, Canada, September 22-25, 2005.
B
This study was supported by the Programme Algan, Porto, Portugal,
European Union Programme of High Level Scholarships for Latin America,
identification no. E03D17358CL.
* Corresponding author. Tel.: +353 1 4096420; fax: +353 1 4550201.
E-mail address: prem.puri@ucd.ie (P. Puri).
Index words:
Porcine colon;
Distal colon;
Whole-mount
preparation;
Nitrergic innervation;
Ganglia density;
Ganglion cell number;
Ganglion cells size;
Enteric nervous system
Journal of Pediatric Surgery (2006) 41, 1029 – 1035
www.elsevier.com/locate/jpedsurg
Meier-Ruge [1] in 1971 described a malformation of the
enteric plexus that clinically resembled Hirschsprung’s
disease, called intestinal neuronal dysplasia (IND). Intestinal
neuronal dysplasia is characterized by the presence of giant
ganglia in the submucosal plexus, enlarged parasympathetic
nerve fibers in the submucosa, and increased acetylcholines-
terase activity in the mucosa [2,3]. However, the criteria for
diagnosis of IND remain controversial, and several authors
have raised doubts about its existence as a distinct histopath-
ological entity and its real correlation with clinical symptoms
[4,5]. In particular, it has been suggested that proposed
diagnostic criteria relating to ganglion cell density may
overlap with age-related changes [2,3]. An important reason
for this confusion is insufficient knowledge of the morphol-
ogy of the normal enteric nervous system through life.
Nitric oxide is the most important inhibitory neurotrans-
mitter in the gastrointestinal tract in various species and can be
clearly shown by nicotinamide adenine dinucleotide phos-
phate diaphorase (NADPH-d) histochemistry [6,7].Whole-
mount preparation is an elegant technique for visualization of
the myenteric and submucosal plexuses. It provides a method
for the detailed study of the 3-dimensional morphology of the
meshwork of nerves and neurons and, therefore, is far superior
to standard tissue sections in the investigation of the enteric
nervous system [6]. NADPH diaphorase–positive ganglion
cells have been estimated in whole-mount preparations to
represent about 34% of all neurons in the human myenteric
plexus [8]. Because the organization of the porcine enteric
nervous system possesses functional and histologic similarities
with the human one, the pig intestine is the most suitable
experimental model for studying the human enteric nervous
system [9,10].
The aim of this study was to investigate the normal
nitrergic ganglia density, neuron number and morphology in
whole-mount preparations of the submucosal plexus of the
porcine distal bowel from fetal life to adulthood.
1. Materials and methods
1.1. Tissue sampling and whole-mount preparation
Bowel specimens were obtained from porcine fetuses
of gestational age 60 days (n = 5) and 90 days (n = 5) and
1-day-old piglets (n = 5), 4-week-old piglets (n = 5),
12-week-old piglets (n = 5), and 1-year-old adult pigs (n =
5). The animals were provided from the Institute of
Experimental Clinical Research, Skejby Sygeh, University
of Aarhus, Denmark. The study was approved by the Danish
authorities of animal protection, permission no. 200
601-068. The large bowel was removed from the piglets
and placed in 4% paraformaldehyde in 0.1 mol/L phos-
phate-buffered saline (PBS) for 48 hours at 48C. Afterward,
specimens were transferred into sterile containers filled
with PBS and stored at 48C until further use. The distal
bowel was opened along the mesenteric border and rinsed.
Four samples of distal bowel from each specimen were
cut for further processing. Samples were pinned flat on a
silicone plate, and whole-mount preparations were made
under a dissecting microscope (Leica S8 APO; Heerbrugg,
Switzerland). Initially, the mucosal layer was scraped
out along with the inner submucosal plexus (Meissner’s
plexus) using fine-pointed forceps. After that, the submu-
cosal layer containing the outer submucous plexus (Scha-
badasch or Henle plexus) was carefully lifted from the
muscle layer with a fine forceps starting in one corner of the
tissue sample.
1.2. Staining procedure
For NADPH diaphorase histochemistry, the submucous
laminae were placed in a sterile 12-well plate (Corning,
New York, NY) and were incubated in 2 mL of the staining
solution containing 1 mg/mL b-NADPH (Sigma-Aldrich,
St Louis, MO), 0.25 mg/mL nitrobluetetrazolium (Sigma-
Aldrich), and 0.3% Triton X-100 (BDH Laboratory
Supplies, Dorset, UK) in a 0.05 mol/L TRIS HCl buffer
(pH 7.6) for 2 hours at 378C and then left in the staining
solution overnight at room temperature. After achievement
of the desired staining intensity, specimens were rinsed in
PBS for 15 minutes and then mounted on Polysine
microscope slides (BDH) using Glycergel mounting medi-
um (DakoCytomation, Glostrup, Denmark).
1.3. Morphometry
A ganglia was defined as a group of at least 3 ganglion
cells with a distance between cells not exceeding 2-cell
diameters as previously described [11]. Density of NADPH
diaphorase–positive ganglia was measured by counting the
total number of ganglia per 1 cm
2
under a light microscope
(Leica DMLB) using 200 magnification. For that purpose,
a 1-cm
2
square graticule was drawn on the coverslip of the
newborn, 4-week, 12-week, and adult specimens. Because
E60 and E90 specimens were smaller than 1 cm
2
, a smaller
square graticule was drawn on them and the results were then
converted to 1 cm
2
. The number of NADPH diaphorase–
positive neurons per ganglia was determined by counting
them in 25 adjacent ganglia in each specimen under a light
microscope (Leica DMLB). All profiles of positively stained
cells were identified and counted. In some cell clusters,
identification of cell profiles was confirmed by adjusting the
focal depths of the objective. Size of NADPH diaphorase–
positive neurons was determined by analyzing the photo-
graphs of at least 85 neurons from 10 different ganglia with
clearly delimitated individual neurons from each specimen.
The border of each neuron and its nucleus were marked by
hand in the digitalized image, and the total area of the neuron
and the nucleus was measured using software for image
analysis (Image J 1.5 Beta 1; Research Services Branch,
National Institute of Mental Health, Bethesda, Md, USA).
The area of the cytoplasm was calculated by subtracting the
area of the nucleus from the area of the whole neuronal body.
S. Montedonico et al.1030
1.4. Statistical analysis
All numerical data are expressed as mean FSD. The
normal distribution of each group was assessed with
Kolmogorov-Smirnov test. After that, analysis of vari-
ance test was used to compare ganglia density, number of
ganglion cells per ganglia, and ganglion cell size among the
different age groups, with the Student-Newman-Keuls test
being used for pairwise comparisons. A Pvalue of less than
.05 was considered statistically significant. All statistical
tests were performed using a commercially available
software package (SPSS 11.0 Statistical Analysis Software,
Chicago, IL).
2. Results
Whole-mount preparations of the submucosal plexus
facilitated visualization of a regular mesh of nerve bun-
dles with ganglia containing NADPH diaphorase–positive
ganglion cells at the intersections. The ganglia were clearly
separated one from another, and no neurons were seen along
the bundles. Staining of NADPH diaphorase–positive
neurons within each ganglia was not uniform. About one third
of the neurons showed very strong staining, whereas the re-
maining neurons were moderately or weakly stained (Fig. 1).
2.1. Ganglia density in different age groups
The gross morphology of the submucous plexus varied
with age. The meshwork became progressively less dense
with increasing age. The highest number of ganglia per
square centimeter was found at E60 (1912.5 F279.69
ganglia per square centimeter). After that period, ganglia
density fell markedly and constantly with age until adulthood
(E90, 775 F85.79 ganglia per square centimeter; newborn,
412.5 F50.51 ganglia per square centimeter; 4 weeks
old, 264.28 F28.78 ganglia per square centimeter; 12 weeks
old, 71.33 F18.14 ganglia per square centimeter; adult,
29.8 F5.61 ganglia per square centimeter; values in each
age group are statistically different from the previous and the
following age group, Pb.001) (Fig. 2).
Fig. 2 Ganglia density in the submucosal plexus of the distal pig colon at different ages (logarithmic transformation). Ganglia density
decreases progressively with age (mean FSD). E60 indicates pig fetus of gestational age 60 days; E90, pig fetus of gestational age 90 days;
newborn, newborn piglet; 4 weeks, 4-week-old piglet; 12 weeks, 12-week-old piglet; adult, 1-year-old adult pig. *Pb.001.
Fig. 1 Whole-mount preparations of the submucosal plexus of the distal pig colon. A, A mesh of nerve bundles with ganglia containing
NADPH-d–positive ganglion cells at the intersections is clearly seen (original magnification 100). B, Typical NADPH-d–positive
submucosal ganglia (original magnification 200).
Developmental changes in submucosal nitrergic neurons in the porcine distal colon 1031
2.2. Number of ganglion cells per ganglia in
different age groups
The number of NADPH diaphorase–positive ganglion
cells per ganglia increased significantly during the intra-
uterine life until the piglets were born (E60, 16.33 F7.79;
E90, 23.41 F9.96; newborn, 30.29 F13.37; values in each
age group are statistically different from the previous and
the following age group, Pb.001). From the newborn
period until the piglets were 4 weeks old, the mean number
of neurons per ganglia decreased significantly (30.29 F
13.37 for newborn vs 18.89 F10.61 for 4 weeks old, Pb
.001). The number of ganglion cells per ganglia remained
constant from 4 weeks until adulthood (4 weeks old, 18.89
F10.61; 12 weeks old, 17.51 F10.55; adult, 17.62 F8.67;
P= not significant). In the adult pig, the mean number of
NADPH diaphorase–positive ganglion cells was almost the
same that in E60 piglets (Fig. 3).
2.3. Ganglion cell size in different age groups
A marked increase in the size of NADPH diaphorase–
positive neurons was seen with increasing age (E60, 71.91
F14.67 l
2
; E90, 102.51 F28.52 l
2
; newborn, 157.21 F
48.02 l
2
; 4 weeks old, 239.94 F88.81 l
2
; 12 weeks old,
257.19 F102.45 l
2
; adult, 392.51 F194.71 l
2
; values in
each age group are statistically different from the previous
and the following age group, Pb.001). However, the
increase in cell size was found to be predominantly caused
by an increase in cytoplasm (E60 cytoplasm area, 28.71 F
5.11 l
2
; E60 nucleus area, 43.2 F9.56 l
2
; E90 cytoplasm
area, 51.23 F15.23 l
2
; E90 nucleus area, 51.28 F13.29 l
2
;
Fig. 4 Ganglion cells size in the submucosal plexus of the distal pig colon at different ages. A marked increase in ganglion cells size is seen
with increasing age (mean FSD). However, this increase is predominantly because of the growth of the cytoplasm. *Pb.001.
Fig. 3 Number of NADPH diaphorase–positive ganglion cells per ganglia in the submucosal plexus of the distal pig colon at different ages
(mean FSD). Note that the number of ganglion cells per ganglia increase significantly during the fetal life. After the newborn period, there is
a marked reduction in the number of ganglion cells per ganglia, remaining constant thereafter. *Pb.001.
S. Montedonico et al.1032
newborn cytoplasm area, 88.74 F32.99 l
2
; newborn
nucleus area, 68.47 F15.03 l
2
; 4-week-old cytoplasm
area, 158.71 F64.81 l
2
; 4-week-old nucleus area, 81.23 F
24.00 l
2
; 12-week-old cytoplasm area, 174.81 F77.67 l
2
;
12-week-old nucleus area, 82.38 F24.78 l
2
; adult
cytoplasm area, 279.89 F162.14 l
2
; adult nucleus area,
112.62 F32.57 l
2
; values in each age group are statistically
different from the previous and the following age group,
Pb.001) (Figs. 4 and 5).
3. Discussion
Our results show that significant morphological changes
occur in the submucosal plexus of the porcine distal colon
throughout life. We found an inverse relationship between
ganglia density and ganglion cells size with age. Ganglia
density decreased progressively with gestation and postna-
tal age, whereas ganglion cells size increased with age. On
the other hand, the mean number of ganglion cells per
ganglia showed a distinct pattern reaching the highest value
at the neonatal period and decreasing thereafter to stabilize
at 4 weeks in the pig. Comprehensive information regarding
the prenatal and postnatal normal morphological changes of
the enteric nervous system is scanty and is extremely
important when interpreting histopathological findings in
early childhood. The gold standard test when evaluating a
child with chronic constipation and obstructive symptoms is
a suction rectal biopsy, which normally comprises mucosa
and submucosal layers of the rectum. Hirschsprung’s
disease, IND, and other dysganglionosis may be diagnosed
by evaluating the submucosal innervation pattern. For this
reason, our study focused on the submucosal plexus of the
distal colon. We used whole-mount preparation technique,
which produces a 3-dimensional picture, to better show the
structure of neuronal networks and their relationship of
branching and interconnecting nerve fibers to each other
and to the neighboring tissues [6]. Quantitative morpho-
logical analysis of the enteric plexuses are therefore much
more accurate with whole-mount technique than with
standard tissue sections, which only partially show the
morphology of the plexus and whose results may vary
depending on the number and thickness of sections studied
[6]. We chose the pig as our experimental model because it
is a large mammal with an enteric nervous system that
possesses striking similarities with the human one [9,10].
Nitric oxide is an important inhibitory neurotransmitter that
mediates relaxation of the smooth muscle of the gastroin-
testinal tract [7]. The enzymes involved in the neuronal
generation of nitric oxide are the constitutive neuronal
isoform of nitric oxide synthase and NADPH diaphorase
[6]. A one-to-one correlation between the 2 enzymes
responsible for nitric oxide synthesis has been found in
the enteric neurons. Consequently, neurons producing nitric
oxide can be detected by using either nitric oxide synthase
immunohistochemistry or NADPH-d histochemistry. It
has been demonstrated that NADPH diaphorase–positive
neurons account for 34% of the total number of enteric
neurons [8]. NADPH-d–positive neurons of the porcine
submucous plexus showed a gradation of reactivity for the
enzyme. Other authors have previously shown similar
findings [12,13].
Fig. 5 Ganglion cells in the submucosal plexus of the distal pig colon at different ages. Ganglion cells increase their size throughout life
predominantly because of an increase in cytoplasm. All photographs are taken with the same magnification (original magnification 400).
Developmental changes in submucosal nitrergic neurons in the porcine distal colon 1033
We found the meshwork to become progressively and
markedly less dense with increasing age, which was
objectively assessed by quantifying the number of ganglia
per surface area. Ganglia density significantly changed from
one age point to the following one until the adulthood. This
is a known phenomenon that has been studied in different
species including human and is known to occur in both
myenteric and submucosal enteric plexuses and also in the
bladder [8,11,13-18]. Growth of the bowel with increasing
surface area probably causes the originally densely packed
network to expand and leads to a lower ganglia density with
increasing age.
We were surprised by our findings on the variation of
the number of ganglion cells per ganglia throughout life.
They significantly increased during the intrauterine life
until the piglets were born. Then, from the newborn period
until the piglets were 4 weeks old, the number of neurons
per ganglia decreased significantly, remaining constant after
that until adulthood, when the number of ganglion cells per
ganglia return to be the same as during early intrauterine
life. If we consider that pig natural life span is approxi-
mately 17 years and they reach puberty at 7 months old, a
4-week-old piglet would be the equivalent to a 2-year-old
infant [19]. If we extrapolate our findings to the human
situation, that would mean that the number of ganglion cells
per ganglia decreases progressively after birth until the
child is 2 years old to remain constant thereafter. Similar
findings have been shown in the chicken myenteric plexus,
whose ganglion cells per ganglia increase around the
perinatal period [17,18]. Coerdt et al [15] have recently
studied sections of human colonic submucosal plexus.
Their results are strikingly similar to the present study.
They found the mean number of ganglion cells per ganglia
to be highest in the group with a gestational age of less than
35 weeks, followed by the group of patients aged less than
1 year, and slightly decreasing thereafter. It is intriguing
why neurons increase their number during fetal life to
decrease then after birth. We speculate that this could be an
adaptation phenomenon of the enteric plexuses to the
greater demand for innervation at birth, when the actual
bowel function starts. However, this may also be a purely
developmental phenomenon.
Our measurements revealed an increase in ganglion cells
size during life from relatively small cells with small
cytoplasm in the early fetal period to large cells with
enlarged cytoplasm in the adult. This finding has been
previously reported in the human enteric nervous sys-
tem [8,16,20] and also in the pig bladder [13] and has been
considered a sign of maturation of the neurons. The
increase in cell size is explained by the growth of the
bowel: an increase in volume of an innervated organ is
normally accompanied by an increase in the volume of
intramural nervous tissue and particularly in the cytoplasm.
Moreover, it has been demonstrated that neurons react upon
an increase of muscle mass by increasing their size even in
adult rats [21].
The present findings suggest that enteric nervous system
is dynamic and developmental changes occur all throughout
life but especially in the perinatal period and during the first
years of life. Applied to the clinical setting, it implies that
interpretation of enteric nervous system pathology is highly
dependent on age of the patient. Intestinal neuronal
dysplasia is characterized by the presence of hyperganglio-
nosis and giant ganglia with more than 7 ganglion cells in
the submucosa [2,3]. Our present findings may have
implications for the understanding of pathophysiology of
IND. We hypothesize that IND is a developmental
maturation phenomenon, with histopathologic features of
IND corresponding to the normal enteric plexus findings in
the late gestational period. Further evidence supporting this
hypothesis is that although IND is a recognized histopath-
ological and clinical entity [2,3,22], most patients do well
with only conservative treatment [23].
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