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A Possible Association Between Interstitial Cells of Cajal and Neuronal Nitric Oxide Synthase in Infantile Hypertrophic Pyloric Stenosis

Christina Panteli¹, Antonios Filippopoulos¹, Eleni Vrettou², Konstantinos Kallergis¹, Athanasios Zavitsanakis¹
¹1st Department of Paediatric Surgery, Aristotle University of Thessaloniki, Thessaloniki, Greece
²Department of Pathology, Aristotle University of Thessaloniki, Thessaloniki, Greece

 

 

Correspondence

Christina Panteli
Department of Paediatric Surgery
Norfolk and Norwich University Hospital
Colney lane; Norwich NR47UY
tel. 0044 1603 286355
email: This email address is being protected from spambots. You need JavaScript enabled to view it.

 

Abstract

Aim: Despite numerous hypotheses, the pathogenesis of infantile hypertrophic pyloric stenosis (IHPS) remains unknown. Interstitial cells of Cajal (ICC) serve as pacemakers and neurotransmission mediators along the gastrointestinal tract. Nitric oxide (NO) is the primary inhibitory nonadrenergic - noncholinergic neurotransmitter of the enteric nervous system. ICC and NO have been studied separately with regards to their possible role in the pathogenesis of IHPS. The aim of this study was to explore the association between ICC and NO in the normal pylorus and in IHPS.

Material and methods: Specimens obtained from 19 patients with IHPS and 2 control specimens were immunohistochemically stained with antibodies to c-kit and neuronal Nitric Oxide Synthase (nNOS) for ICC and NO respectively and a semiquantitative analysis was carried out. 

Results: Control specimens showed strong immunopositivity for both c-kit positive ICC and nNOS. ICC were markedly reduced in IHPS, or absent. The expression of nNOS was moderately or significantly reduced in the majority of specimens in IHPS. The correlation between c-kit immunoreactivity and nNOS staining intensity was statistically significant. 

Conclusions: Reduced expression of both c-kit positive ICC and nNOS may suggest an interaction between ICC and NO, either by ICC mediating inhibitory neurotransmission in the pylorus or by NO acting as a survival factor for ICC. The association between ICC and NO may have a key role in the pathogenesis of IHPS.

Key words: pyloric stenosis, Interstitial Cells of Cajal, Nitric Oxide

  

Introduction

Infantile hypertrophic pyloric stenosis (IHPS) is the most common disease requiring surgical treatment in the first few weeks of life. Although IHPS has been recognised as a clinical entity since 1888 and the first successful pyloromyotomy was performed in 1911, its pathogenesis remains unclear.

Several theories have been formulated concerning the aetiology of pyloric stenosis including genetic and environmental factors, abnormalities of hormonal control, pyloric innervation, interstitial cells of Cajal (ICC), growth factors, smooth muscle cells and extracellular matrix proteins. Amongst other elements of pyloric innervation, lack of nitric oxide (NO) has been implicated in the pathogenesis of the disease [1]. Interstitial cells of Cajal (ICC) are nonneuronal cells that form networks alongside the enteric nervous system and serve as electrical pacemakers and mediators of motor neurotransmission in the gastrointestinal tract.

ICC express c-kit, a transmembrane protein kinase receptor essential for their development and maintenance. In patients with IHPS, a lack or a significant reduction of ICC in the pyloric muscle has been reported, suggesting a disruption of their network that may account for pyloric dysmotility [2-5]. NO is a major non adrenergic non cholinergic neurotransmitter in the enteric nervous system that mediates pyloric relaxation. The production of NO from L-arginine is catalyzed by neuronal nitric oxide synthase (nNOS). In IHPS, nNOS has been reported to be absent or markedly reduced. Lack or depletion of nNOS may result in failure of the pylorus to relax and therefore pyloric stenosis [6-10]. ICC and NO have been so far studied separately with regards to their role in the pathogenesis of IHPS. The objective of this work was to investigate the degrees of expression of ckit and nNOS in IHPS and in the non diseased pylorus in view of any possible association between ICC and NO.

Material and methods 

After obtaining Institutional Ethics Commitee approval full thickness muscle biopsy specimens were taken at pyloromyotomy from 19 infants (age range 25-60 days) with IHPS and two controls (aged 3 and 11 months) without pyloric disease, at autopsy. Specimens were fixed in 10% neutral buffered formalin solution. After fixation tissue blocks were embedded in paraffin and subsequently 4 μm sections were cut and affixed onto slides. Sections were deparaffined with xylene, rehydrated through descending alcohol solutions and rinsed in tap water. In order to block endogenous peroxidase activity, sections were immersed in 0.5% H2O2 in methanol. Subsequently, consecutive sections were incubated with the primary rabbit polyclonal antibody anti-human c-kit (DAKO, Japan), diluted 1:50 or the primary mouse monoclonal antibody anti-human nNOS (Novocastra, United Kingdom), diluted 1:40 for 60 minutes at room temperature. Slides were then rinsed with TBS (Tris Buffered Saline, pH 7.6) and the secondary antibodies (biotinylated anti-rabbit and anti-mouse IgG respectively) were applied. Sections were incubated in ABC (Avidin-Biotin Complex System) followed by DAB (3,3’ - Diaminobenzidine). After washing with tap water, the sections were counterstained with hematoxylin. A semiquantitative analysis was carried out and the ICC and nNOS expressions were classified into four grades. The density of immunostaining was graded as follows: 0, absence of positive staining; 1, positive staining 10%; 2, positive staining 10%-50%; 3, positive staining > 50%. Grading refers to the total staining in the myenteric plexus and the muscle layers. The SPSS 16.0 Statistical Software was used for data analysis. Clinical data are expressed as median (range). The Spearman correlation test was used to analyze the relation between ICC and nNOS expressions. A p value of < 0.05 was considered statistically significant.

Results

Sixteen boys and 3 girls were analysed. Median gestational age was 38 weeks (range 36-40) and median duration of symptoms was 12.5 days (range 1-40); at operation the median age was 38 days (range 25-60) while the postconceptual age was 44 weeks (range 40-48). Semiquantitative analysis of staining for c-kit and nNOS revealed grade 3 immunostaining for both controls. In IHPS specimens c-kit immunoreactivity was not demonstrated in 6 patients (grade 0) whereas staining was weak in 12 patients (grade 1) and moderate in 1 patient (grade 2). Strong staining for c-kit (grade 3) was not observed in any specimen ( fig. 1).

cajal 1

Figure 1. Grade 1 (a) and Grade 2 (b) c-kit immunoreactivity in IHPS

Immunoreactivity for nNOS was absent in 2 patients (grade 0), weak in 6 patients (grade 1), moderate in 8 patients (grade 2) and intense in 3 patients (grade 3), ( fig. 2). All specimens with negative c-kit immunoreactivity demonstrated either absent or weak nNOS staining. In specimens with positive staining for both c-kit and nNOS, nNOS expression was stronger in comparison to c-kit expression. The degree of expression for c-kit was not significantly correlated with gestational age (p=0.09), postconceptual age (p=0.18), age at operation (p=0.62) or duration of symptoms (p=0.94). Similarly, nNOS staining density was not correlated with gestational age (p=0.25), postconceptual age (p=0.98), age at operation (p=0.49) or duration of symptoms (p=0.75). Using Spearman correlation for linear regression, the correlation between c-kit immunoreactivity and nNOS staining intensity was statistically significant at p < 0.01, with a correlation coefficient of r =0.805 ( fig. 3). 

cjal 2

Figure 2. Grade 1 (a) and Grade 2 (b) nNOS immunoreactivity in IHPS 

cajal 3

Figure 3. Correlation between c-kit and nNOS expressions in IHPS patients

Discussion

ICC and NO have been studied with regards to their role in the pathogenesis of IHPS and have been found to be either absent or reduced in the hypertrophied pyloric muscle. Similar findings have been reported in a number of intestinal motility disorders [11-14].

The pacemaker activity of ICC is well described. ICC trigger the generation of spontaneous pacemaker currents, known as slow waves, which are essential for effective peristalsis [14]. The involvement of ICC in IHPS has been mainly attributed to this particular function suggesting that lack of ICC and therefore disruption in slow waves contributes to pyloric dysmotility [3, 5]. Recent evidence suggests that the electric activity is provided by a subpopulation of ICC, the myenteric ICC, whereas a different subpopulation, the intramuscular ICC, mediate motor innervation including nitrergic inhibitory neurotransmission [15]. NO released on stimulation of motor enteric neurons mediates smooth muscle relaxation along the gastrointestinal tract [11]. Impaired NO-induced pyloric relaxation has been postulated to play a key role in IHPS [7-10]. The results of the present study are in agreement with previously reported studies [2-10]. ICC are either absent or markedly reduced in patients with IHPS. nNOS expression is absent or low in the hypertrophied pylorus in the majority of patients whilst strong immunostaining was noted in a small number of patients. The observation of strong nNOS immunoreactivity in a subset of patients with IHPS has been previously reported [16].

A remarkable finding of this study is the quantitative association between the degrees of immunoreactivity for c-kit and nNOS in IHPS. This finding raises the question whether there is a causal relationship between ICC and nNOS expressions. There is evidence on three possible ways of interaction between ICC and nitrergic innervation [17-19]. ICC and nitrergic neurons may be interdependent, each being essential for the development and maintenance of the other cell type, or ICC alterations may affect nitrergic neurotransmission or vice versa. Studies on genetic models have shown that functional ICC develop in the absence of enteric neurons [20] and conversely, in the absence of intramuscular ICC the nitrergic network appears to be intact [21].

With regards to the role of ICC on nitrergic innervation, it has been suggested that ICC may produce NO and thus, amplify inhibitory neurotransmission in the enteric nervous system [22]. The classic concept of neuromuscular neurotransmission in the gastrointestinal tract has been that neurotransmitters are released from nerve endings and activate responses in neighbouring smooth muscle cells via binding to specific receptors expressed by smooth muscle cells. However, recent studies have suggested that intramuscular ICC are interposed between nerve terminals and smooth muscle cells and form close contacts with both types of cells. Abundant specialized junctions exist between nerve terminals and ICC whereas similar structures between nerve terminals and smooth muscle cells are rare. Neurotransmitters released from motor enteric neurons primarily bind to receptors expressed by ICC and the electrical responses generated are conducted to adjacent smooth muscle cells [15, 18]. Genetic animal models have provided important evidence on the role of ICC in nitrergic neurotransmission. In animals lacking intramuscular ICC, NO-dependent inhibitory neuroregulation is either absent or greatly attenuated in vitro [17, 21]. This finding has been challenged by in vivo and in vitro studies [23-25].

Although the function of NO as a neuromodulator in the central nervous system and in smooth muscle relaxation is well recognised, there is accumulated evidence that it is involved in various physiological and pathological events. NO is a cytotoxic free radical that can induce cell death at high concentrations whilst at lower concentrations it appears to have a cytoprotective role [26]. Recently, the effect of loss of nNOS on ICC was studied in mice with targeted disruption of the nNOS gene [19]. These animals serve as the first genetic model for IHPS and, with the exception of gross enlargement of the stomach and hypertrophy of the circular muscle of the stomach and pylorus, display no other abnormalities [27]. The number and volume of ICC in the gastric body of knockout mice was significantly lower compared to tissues from controls but increased following culture with a NO donor, suggesting that NO modulates ICC numbers and volume, possibly by acting as a cytoprotective molecule for ICC [19].

The possible association between ICC and nitrergic neurotransmission has been studied in motility disorders in adults, such as diabetic gastroparesis and achalasia. Decreased numbers of ICC and low expression of nNOS are seen in diabetic gastroparesis [28]. In gastric biopsies from diabetic patients a substantial decrease in both ICC and nNOS densities has been observed; the extent of c-kit and nNOS colocalisation was lesser in diabetic patients in comparison to controls implying a possible link between impaired nitrergic inhibitory neurotransmission and loss of ICC. The results from studies on patients with achalasia are conflicting. One study showed significant ICC reduction that was independent of the extent of nitrergic nerve degeneration [29]. Another study found that c-kit immunopositivity was linearly related to nNOS staining intensity in oesophageal specimens from patients with achalasia and the correlation was statistically significant [30].

The issue of c-kit expression in relation to nNOS immunoreactivity in IHPS has not been previously explored. The significant correlation between ICC reduction and nNOS impairment demonstrated in this study raises the possibility of a causal relationship between ICC and NO in the pathogenesis of IHPS. A primary deficiency in ICC might be responsible for nNOS reduction resulting in impaired pyloric relaxation and therefore IHPS or a primary lack of nNOS might be responsible for a reduction in ICC resulting in attenuated neurotransmission and IHPS. Although at present nNOS reduction underlying depletion of ICC appears more likely, the question of the primary disorder remains [19, 25]. The results of this work must be interpreted considering its limitations. First, obtaining matched controls for IHPS poses a challenge, as sampling during laparotomy for other pathology is rarely justified whereas the vast majority of autopsy specimens are unsuitable for immunostaining. Second, a statistical correlation between ICC and NO does not necessarily indicate a causal relationship. Third, a whole mount preparation with double labelling for c-kit and nNOs and confocal electron microscopy would probably provide more accurate and reliable results. Therefore, further studies are needed to define the relation between ICC and NO with regards to the pathogenesis of IHPS.

In conclusion, the present study confirms that both ICC and NO mediated inhibition are either absent or markedly reduced in the majority of patients with IHPS. The degrees of c-kit and nNOS expression are correlated in a linear mode, suggesting that in the pathogenesis of IHPS, the association between ICC and nitrergic innervation, rather than ICC or NO separately, might have a key role. 

 

 

 

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