Download The role of nitric oxide synthase in infantile hypertrophic pyloric

Jornal de Pediatria - Vol. 77, Nº4, 2001 307
0021-7557/01/77-04/307
Jornal de Pediatria
Copyright © 2001 by Sociedade Brasileira de Pediatria
ORIGINAL ARTICLE
The role of nitric oxide synthase
in infantile hypertrophic pyloric stenosis
Irnak M. Barbosa,1 Saulo M.R. Ferrante,2 Carlos A. Mandarim-de-Lacerda3
Abstract
Objective: to experimentally reproduce, in rats, the findings corresponding to the histopathology of
infantile hypertrophic pyloric stenosis (IHPS), using nitric oxide synthase (NOS) inhibitor (L-NAME).
Methods: L-NAME was administered to pregnant rats (L-NAME group), from the 14th gestational day
on in order to reproduce the model of NOS inhibition in the production of IHPS. This group was then
compared to control animals. After birth, all the animals in the L-NAME group were maintained under NOS
inhibition until the 42nd day of life, when they were sacrificed. The control animals, which did not receive
any kind of drug, were also sacrificed on the 42nd day of life. The animals and their internal organs were
analyzed and weighed. The pyloric region was technically prepared and observed through light microscopy.
Results: the L-NAME group presented lower body and intestinal weight and higher gastric weight than
the control group. Light microscopy revealed hypertrophy of the circular smooth muscle layer of the pyloric
muscle in L-NAME animals.
Conclusions: this work reproduced an experimental model of an IHPS study, confirming the effect of
NOS blockade on the pyloric musculature.
J Pediatr (Rio J) 2001; 77 (4): 307-12: pyloric stenosis, nitric oxide synthase, L-NAME.
Introduction
Infantile hypertrophic pyloric stenosis (IHPS) is a
common disorder with an incidence of 0.3% in newborn
infants. It occurs mainly from the third to the sixth week of
life, and is characterized by hypertrophic circular pyloric
muscle and by developing with pyloric obstruction and
persistent vomiting.
Until the present moment, the etiology of IHPS is not
fully understood. It is believed, however, that it involves an
association of hereditary and environmental factors. Many
hypotheses have been presented involving pyloric muscle
innervation, hormone control, extracellular matrix proteins,
peptidergic and nitrergic innervation, and, more recently,
smooth muscle growth factor alterations.1,2 Currently, it is
understood that primary abnormalities of innervation of
enteric nerves, associated with absence of nitric oxide
synthase (NOS) and interstitial cells of Cajal,3 are more
promising theories for explaining the pathophysiology of
IHPS.
1. Master, Pediatric Surgery.
2. Associate Professor, Pediatric Surgery, Surgery Department, School of
Medicine, Universidade Federal do Rio de Janeiro.
3. Professor, Morphometry Laboratory, Biomedical Center, Universidade
do Estado do Rio de Janeiro.
307
308 Jornal de Pediatria - Vol. 77, Nº4, 2001
Recent studies have shown that nitric oxide (NO) is an
unstable substance, synthesized from L-arginine by NO
synthase (NOS),4 and that it is one of the main relaxant
transmitters of gastrointestinal smooth muscle. The neuronal
nitric oxide synthase is found in both the central nervous
system and in the peripheral nervous system.5 The activity
of NOS in the gastrointestinal tract has been detected by
immunohistochemistry in the myenteric plexus and in muscle
planes (circular and cross-sectional). Huang et al.6 were
able to generate mutant mice by homologous recombination
with alteration of the locus of the NOS gene. The mice
presented lack of NOS activity and grossly enlarged stomachs
with hypertrophy of the circular muscle layer.7
The objective of our study is to experimentally reproduce
the histopathological findings corresponding to IHPS in
cases of inhibition of NOS.
Materials and methods
We used Wistar rats from the Fiocruz central animal
facilities, state or Rio de Janeiro, Brazil. Three pregnant
females, one with 14 days of gestation and two with 16 days
of gestation, were named D14 and D16 (NOS inhibitor
group) and D16c (control). Until the end of gestation, the
rats remained at the animal facilities of the Morphometry
Laboratory of the Universidade Estadual do Rio de Janeiro
(UERJ). The facilities were controlled for temperature,
lighting, and humidity. Rats were fed with appropriate feed
(Novilab TM) and given potable water ad libitum.
The role of nitric oxide synthase... - Barbosa IM et alii
10% at room temperature for 48 hours. Subsequently,
fragments were embedded in paraffin wax and divided into
sections of 3 micrometers with 10 micrometers of thickness.
Sections were prepared and stained by hematoxylin-eosin
and trichromic (Masson).
We calculated descriptive statistics. Differences between
groups were tested using the nonparametric Mann-Whitney
test. Significance was considered for P = 0.05.
Results
Groups were assessed according to weight of pups.
Comparison of weight on the 21st day of life of L-NAME
groups (D14 and D16) and controls (D16c) was not
significant (Figure 1). On the 42nd day, however, this
comparison indicated a significant difference on weight
gain between the experimental (L-NAME) and control
groups (Figure 2). We observed values for weight and
dimension of bodily structures removed from the 18 pups.
Average ± standard deviation of length of small intestines
and of weight of the stomach were, respectively 90.72 ±
5.89 cm and 1.50 ± 0.27 g. In the L-NAME groups (D14,
D16), the average stomach weight was higher than in the
control group.
From the beginning of the study until the end of gestation,
rats D14 and D16 received nitric oxide synthase inhibitor LNAME (N(G)-nitro-L-arginine methyl ester hydrochloride;
Sigma Chemical Co.; St. Louis; 98H1427) at 50 mg/kg/day.
The inhibitor was diluted in drinking water administered at
sufficient minimal daily amounts. There was no significant
difference on the volume intake of animals. Rat D16c was
not administered L-Name (control).
D14, D16, and D16c litter sizes were of, respectively,
seven, six, and five pups. On the 21st day of life, pups were
removed from their mothers, weighed, followed-up for
another 21 days, and then sacrificed. After weaning, all
pups were fed with the same diet fed to their respective
mothers until weaning. L-NAME was administered to D14
and D16 pups, but not D16c, with drinking water for another
21 days and at 50 mg/kg/day.
At 42 days of life, animals were sacrificed by inhalation
of sulfuric ether and intracardial injection of approximately
0.5 ml of KCl. We removed the stomach, the pylorum, and
the small intestines, which were all measured and weighed.
The intestines (from the duodenum to the ileocecal valve)
were removed, rid of intestinal material (cleaned), and
measured. Next, fragments from the whole extension of the
wall of the pyloric region were fixed in buffered formalin at
Figure 1 - Bar graph (mean±standard deviation) showing body
weight on the 21st day after birth for D16 (L-NAME)
and D16c (control) groups
The difference in the comparison of stomach weight to
body weight of the L-NAME and control groups was
statistically significant (Figure 3). The average intestine
weight was 7.20 ± 0.87 g. The difference in the relation
weight-to-small intestine length of the experimental and
control groups was also statistically significant (Figure 4).
The role of nitric oxide synthase... - Barbosa IM et alii
Jornal de Pediatria - Vol. 77, Nº4, 2001 309
with IHPS both quantitative and qualitatively. This
discussion was initiated by Belding and Kernohan, 8 who
posited that patients with IHPS presented a decrease in the
number of ganglial cells and neural fibers as a result of a
degenerative process. These findings were later confirmed
by Sptiz and Kaufmann.9
Friesen and Boley,10 following a comparative analysis
of neural cells of IHPS children and normal fetuses, suggested
that IHPS patients present immaturity of ganglial cells.
Later on, others observed abnormalities of nerve terminations
of the pylorum11 and of nerve supporting cells,12 in addition
to ultrastructure abnormalities of the myenteric plexus and
pyloric muscle.
Figure 2 - Bar graph (mean±standard deviation) showing body
weight on the 42nd day after birth for D16 (LNAME) and D16c (control) groups
Histological examination of circular pyloric muscle of
the 13 pups that received L-NAME indicated a statistically
significant difference in relation to thickness of the pylorum.
Cross-sections indicated that hypertrophy of the circular
muscle layer of the pylorum presented the largest diameter,
which was twofold the thickness of pylorum in controls
(Figure 5).
Discussion
The etiology of IHPS is still not fully understood despite
the advancements in molecular biology and genetic
engineering. More recent studies have indicated
abnormalities of the enteric nervous system (ENS) in patients
Table 1 -
Figure 3 - Bar graph (mean±standard deviation) showing the
stomach/body weight ratio for sacrificed rats in D16
(L-NAME) and D16c (control) groups
Average weight of pups and of their stomach and intestine
Variable
D14 (n=7)
D16 (n=6)
D16c (n=5)
P‡
Weight (±SD*,g†) on the 21st day
29±2.62
53.6±4.26
59.3±5.35
0.06
Weight (±SD,g) on the 42nd day
77.4±9.83
144.6±23.07
168.8±27.52
0.04
Weight of small intestine (±SD,g)
6.504±0.84
6.035±0.28
8.160±0.24
0.006
Weight of stomach (±SD,g)
1.504±0.27
1.691±0.20
1.250±0.08
0.006
* standard deviation; †: grams; ‡: significance level (P< 0.05), nonparametric Mann-Whitney test
310 Jornal de Pediatria - Vol. 77, Nº4, 2001
The role of nitric oxide synthase... - Barbosa IM et alii
showed that there is a synergic activity between VIP and
NO with in vitro analyses of smooth muscles fibers of the
pylorum. The author concluded that VIP stimulates
production of NO. In this sense, VIP maintains and amplifies
relaxation of smooth muscle cells and is released
presynaptically by NO stimulation. NO also acts on the
initial stage of relaxation.
Figure 4 - Bar graph (mean±standard deviation) showing the
weight/small intestine length ratio for sacrificed rats
in D16 (L-NAME) and D16c (control) groups
New techniques have allowed for identification of
gastrointestinal peptides and description in better detail of
peptidergic innervation. Malmfors and Sandler1 described
abnormalities of peptidergic innervation by showing
reduction in nerve fibers specific for the vasoactive intestinal
peptide (VIP), substance P, and enkephalin in IHPS patients.
VIP is found in high concentrations in the pylorum and is
related to relaxation of gastrointestinal tract smooth
musculature. It is also probably one of the elements
responsible for control of the pyloric sphincter.13 Grider14
Barajas-Lopez15 and Serio16 showed that interstitial
cells of Cajal (ICC) operate as the pacemaker cells regulating
electrical waves in intestinal smooth musculature. Langer 3
observed a decrease in ICC count in IHPS patients. This
finding was confirmed by immunohistochemistry using a
specific antiserum raised against c-kit, a tyrosine kinase
receptor expressed by interstitial cells. 17
NO, initially identified as an endothelium derived
relaxing factor (EDRF),18,19 is known as a powerful
noncolinergic, nonadrenergic neurotransmitter that is
involved in inhibitory innervation of gastrointestinal tract
smooth musculature5,20 IHPS patients have been reported
with lack of NADPH-diaphorase (enzyme considered
identical to NOS in the central and peripheral nervous
systems). Biopsy from pyloric muscle sections from IHPS
children presented decreased NOS in neural fibers of the
circular muscle layer of the pylorum. These biopsies also
indicated that NOS activity was preserved in the myenteric
plexus.21,22 Mutagenic rats (knockout) with lack of NOS
are fertile and present no abnormalities of the CNS. However,
they do present grossly enlarged stomachs with hypertrophy
of the circular muscle layer of the pylorum.6 Kusafuka and
Puri23 showed lower levels of messenger RNA in neuronal
nitric oxide synthase gene in IHPS patients.
Figure 5 - Photomicrographs of the pyloric region (trichromic (Masson) staining, bar: 0.2mm). Transition
between pylorus and duodenum in control animals (A) and in L-NAME animals (B). The arrows
indicate the initial portion of the duodenum (Brünner’s glands)
The role of nitric oxide synthase... - Barbosa IM et alii
The formation of NO increases during gestation, which
explains maternal vasodilatation during this period. The use
of NOS inhibitors at the end of the gestation in rats did not
affect the health of the mother; however, it did affect the
litter with intrauterine growth retardation and ischemic
alterations with hemorrhagic disruptions of hind limbs.25,26
In addition, studies have also observed growth retardation
during the neonatal period in rats administered L-NAME,
which was associated with increase in stomach size, decrease
in weight of intestines, and hypertrophy of the muscular
layer of the pylorum. The comparison of brain weight in
relation to the vitals indicated malnutrition in rats receiving
L-NAME due to pyloric obstruction.26
In our study, we observed that the animals gained weight
from weaning (21st day) to sacrifice (42nd day), which can
be explained by the delayed effect of NOS inhibition and by
the transition from fluid diet (mother’s milk) to solid diet
(feed). The increase in stomach weight and decrease in
small intestine weight, associated with hypertrophy of
circular muscle layer of the pylorum confirmed the findings
of Voelker et al.26
The results of our study corroborate the theory that tries
to explain pathophysiology of IHPS as a consequence of
lack of the nonadrenergic and noncolinergic neurotransmitter
in the pylorum, which Furchgott18 and Ignarro19 described
as being NO. Vanderwinden21 was the first to associate lack
of NOS with IHPS, which was later confirmed by other
authors.6,22,23 The normalization of pyloric innervation
and NOS activity in patients who were submitted to surgical
treatment suggests that lack of NOS in IHPS patients is
transient.
Though further and more specific studies are needed for
assessing the importance of NO in the etiopathogeny of
IHPS, our study suggests that inhibition of NOS, which
leads to loss of relaxation of smooth musculature of the
intestines, is a central element in this process.
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Correspondence:
Dr. Irnak Marcelo Barbosa
Rua General Osório, 109/302
CEP 28625-630 - Nova Friburgo, RJ, Brazil