alexa Mast Cells Connections with Nerve Fibers in Hirschsprung's Disease | Open Access Journals
ISSN: 2157-7099
Journal of Cytology & Histology
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Mast Cells Connections with Nerve Fibers in Hirschsprung's Disease

Hermanowicz A1*, Matuszczak E1, Komarowska M1, Debek W1, Chyczewski L2 and Klek S3

1Department of Pediatric Surgery, Medical University, Bialystok, Poland

2Department of Medical Pathomorphology, Medical University, Bialystok, Poland

3Stanley Dudrick’s Memorial Hospital, General and Oncology Surgery Unit, Skawina, Poland

*Corresponding Author:
Adam Hermanowicz
Department of Pediatric Surgery
Medical University, Bialystok, Poland
Tel: +48857450921
Fax: +48857450920
E-mail: [email protected]

Received Date: October 29, 2013; Accepted Date: December 02, 2013; Published Date: December 04, 2013

Citation: Hermanowicz A, Matuszczak E, Komarowska M, et al. (2013) Mast Cells Connections with Nerve Fibers in Hirschsprung’s Disease J Stock Forex Trad 4:201. doi:10.4172/2157-7099.1000201

Copyright: © 2013 Hermanowicz A. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Introduction: Hirschsprung’s disease (HD) is rare (1/5000 newborns) congenital condition of the bowel nervous system, clinically characterized by dilatation and hypertrophy of colon. There are an increased number of mast cells (MC) in aganglionic bowel segments and in those with intestinal neuronal dysplasia (IND). The exact role of MC in HD is still not known. The aim of this study was to examine the colonic distribution of mast cells (MC) and the relationship between MC and nerve fibers and to estimate connections of these cells with gut nervous system. Material and Methods: Distal colon wall specimens were harvested from patients with normal bowel function and with normal ganglion cells, patients with constipations and with normal ganglion cells and a group of patients with Hirschsprung’s disease. The age of the patients ranged from 2nd day of live to 12 years. The density of the mast cells was calculated using a light microscope with digital camera. The connections of localization of a MC with nerve fibers and ganglions were taken under consideration and observed under light microscope. Test for the significance of differences between groups were performed using the Kruskal-Wallis test. Results: We revealed that there is a greater number of the MC in mucosa, lamina propria, submucosa and muscle layer of large bowel in patients with HD. There was no difference in density of the MC in serosa. Some of the colon MC in HD was located near or they were even attached to the hypertrophic nerve trunks. We found a correlation of MC number with the number of nerve fibers in HD patients. Conclusions: Higher number of MC and their localization in close connection with hypertrophied nerve trunks in patients with HD than in patients with constipation or normal bowel function, is a proof of the influence of MC on defected nervous system in aganglionic bowel. MC probably affect hypertrophied nerve trunks. Mobilization and activation of the mast cells which produce and store NGF can be directed to repair defective nervous system. Obviously, MC repairing reaction is not effective in restoring nervous system function of the gut.

Introduction

Hirschsprung’s disease (HD) is rare (1/5000 newborns) congenital condition of the bowel nervous system, clinically characterized by dilatation and hypertrophy of colon. The true nature of the disease is a aperistaltic, aganglionic part of a distal colon. The main symptoms of the disease are severe bowel obstruction. Harald Hirschsprung in 1886 for the first time described the disease as a “colon dilatation and hypertrophy”. In 1901 Tittel as a first described an absence of nerve ganglions in the colon with Hirschsprung’s disease [1-3].

In HD the aganglionic bowel is characterized by the presence of hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic nerve fibers. All symptoms of HD-delayed meconium passage in newborns, intestinal obstruction, constipation and toxic megacolon are consequences of this fact [1-3]. Kobayashi et al. [4] have found an increased number of mast cells (MC) in aganglionic bowel segments and in those with intestinal neuronal dysplasia (IND). MC are connective tissue cells with cytoplasmic metachromatic granules, involved in many functions in human body. Among a great variety of biological active substances, MC synthesize, store and release nerve growth factor (NGF) responsible for the growth and repair of nerve fibers [4-10]. MC are observed in great amount in digestive tract and also in close contact with blood vessels and nerve fibers [9,11-16]. Substances produced by MC may exert an important effect on embryology, growth, differentiation and regeneration of intestinal nervous system. Additionally, MC products modulate inflammation processes thus influencing on the clinical course of HD [4,9,17]. They are also observed in central nervous system in physiology and pathology. The role of MC as a potential agents for promotion of neoplastic transformation is widely discussed in literature. High mast cells concentration is found in neurofibromas, malignant schwannomas, leiomyosarcomas or in other malignancies of the central nervous system [18]. Although there are a couple of studies on MC in HD in the literature, the exact role of MC in HD is still not known [4,9,12,19-21]. The aim of this study was to examine the colonic distribution of mast cells (MC) and the relationship between MC and nerve fibers and to estimate connections of these cells with gut nervous system.

Materials and Methods

Distal colon wall specimens were harvested from three groups of patients. Group 1: patients with normal bowel function and with normal ganglion cells (n=10), Group 2: patients with severe constipations and with normal ganglion cells (n=12) and Group 3: patients with Hirschsprung’s disease (colon aganglionosis n=20). The full thickness samples were taken transrectally 1-2 cm above dental line (group 2 and 3). In group 1 the colon wall was sampled during surgical repairs performed because of anal atresia. The age of the patients ranged from 2nd day of live to 12 years (mean age 87 months, 49 months and 6 months respectively for groups). The first diagnosis of HD was based on clinical symptoms, radiological findings and rectomanometric results. In every case the final diagnosis of HD was confirmed histologically. The criteria for the recognition of Hirschsprung disease included: an absence of parasympathetic ganglion cells in the myometric and submucosal plexus of the rectum, also extending to the colon and the absence of ganglion cells and the presence of hypertrophic nerve trunks.

None of the patients presented episodes of enterocolitis during or before biopsy. The patients with intestinal neuronal dysplasia were excluded.

The specimens were fixed in a 10% buffered formalin solution, embedded in paraffin at 56°C, then cut into 5 μm slices and stained with hematoxilin and eosin (H+E). Following deparaffinizion, endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol for 30 minutes. The sections were then incubated with the mouse monoclonal antibody for mast cell tryptase (DakoCytomation, Code M 7052) in 1:100 dilutions, for whole night at 4°C temperature and labeled with LSAB plus (DAKO) enzyme reagent and diaminobenzidine (DAB) chromogen for 5 minutes. Sections were counter-stained with hematoxylin. The mast cell tryptase expression is detectible in both resting and activating mast cells.

The density of the mast cells was calculated using a light microscope with digital camera. LUCIA software was used as a morphometric tool. The MC were counted in five random fields in submucosa and muscle layer of the bowel wall using light microscopy (x200). Mucosa and lamina propria were excluded from investigation in this study because of possible impact of a diet on the inflammatory process.

The connections of localization of a MC with nerve fibers and ganglions were taken under consideration and observed under light microscope. Test for the significance of differences between groups were performed using the Kruskal-Wallis test.

The study protocol was approved by local ethics committee.

Results

We have found that MC were localized besides blood vessels and in contact with hypertrophic nerve trunks in aganglionic segment from HD patients. In a bowel specimens from a group 1 the MC were also localized near the nerve fibers and blood vessels but they were also spread in the whole layer (Figures 1 and 2). In the non-HD patients we observed the low concentration of the MC in the colon (Figure 3).

cytology-histology-hirschsprung

Figure 1: Hirschsprung’s disease. Cross-section of the aganglionic colon. Numerous mast cells. Hypertrophic nerve fibers with MC around. (mag 200x).

cytology-histology-disease

Figure 2: Hirschsprung’s disease. Hypertrophic nerve fibers and mast cells localized around (200x mag).

cytology-histology-patient

Figure 3: The non HD patient. Border of the two layers of longitudinal and transverse muscles. Single mast cells. normal ganglia.

The number of mast cells in the submucosa and in the muscle layer was higher in HD patients in comparison with other groups. The difference was seen but it was not statistically significant (p>0.05; p<0.1) in submucosa. In muscle layer the difference was statistically significant (p<0.05). In the muscle layer cells were located solitary and they were in a close connection with nerve trunks and blood vessels.

We evaluated the nerve trunks in 10 High Power Fields. The number of the nerve trunks ranged from 3 up to 10 nerve trunks. All of them showed lack of the ganglion cells, what was confirmed with synaptophysin staining.

Using parameters obtained by image analysis morphometry we found a correlation of MC number with the number of nerve fibers in HD patients (correlation coefficient=0.472). In the non-HD patients the correlation coefficient of MC with number of nerve fibers was 0.382.

We haven’t counted the number of MC connected with a blood vessels but it was also seen that MC were located in perivessel area (Figure 4). This fact if well known in the literature [5,7].

cytology-histology-vessels

Figure 4: Hirschsprung’s disease. MC’s around the vessels penetrating in the muscle layer.

In the non-HD patients the concentration of the MC was lower. The results are in Table 1.

  Control group Constipations Hirschsprung’s disease
Submucosa 95.31 ± 37.41 100.34 ± 74.3 121.32 ± 24.21
Muscle layer 35.16 ± 29.53 24.53 ± 19.17 51.67 ± 36.2

Table 1: Mean number of MC ± standard deviation per mm2 of specimen in submucosa and muscle layer of the large bowel (cells/mm2).

Discussion

The neural hypertrophy in the colonic submucosa associated with aganglionosis is a marker of HD. The thickness of submucosal nerve fibers has been measured by various authors in previous studies [4,16,20]. In this study we analyzed the location and a count of a MC in HD and non-HD cases. MC regulate many different physiological processes. MC through neuronal growth factor (NGF) influence also nervous system [4,9,10,16,21-25]. Some authors, Kobayashi et al., Demirbilek et al., Furgal et al., Puri et al. [4,19,20,25] showed elevated content of NGF and increased number of MC in the aganglionic colon wall in HD in children. In the present study a significant increase was also noticed in MC in HD. We revealed that there is a greater number of the MC in mucosa, lamina propria, submucosa and muscle layer of large bowel in patients with HD. There was no difference in density of the MC in serosa. Furthermore, we also noticed that some of the colon MC in HD were located near or they were even attached to the hypertrophic nerve trunks. We found a correlation of MC number with the number of nerve fibers in HD patients. This is another proof of connection between defected nervous system in aganglionic bowel and MC. MC probably affect hypertrophied nerve trunks. Mobilization and activation of the mast cells can be directed to repair defective nervous system. Obviously, MC repairing reaction is not effective in restoring nervous system function of the gut. Although in HD the MC are probably activated and they are probably producing larger amount of NGF, we still have defective nervous system and all symptoms related with this. So this is certain that NGF produced in MC it is not single substance responsible for repairing a defective nervous gut system [4,9,10,16,25].

Conclusion

Higher number of MC and their localization in close connection with hypertrophied nerve trunks in patients with HD than in patients with constipation or normal bowel function, is a proof of the influence of MC on defected nervous system in aganglionic bowel. MC probably affect hypertrophied nerve trunks. Mobilization and activation of the mast cells which produce and store NGF can be directed to repair defective nervous system. Obviously, MC repairing reaction is not effective in restoring nervous system function of the gut.

We confirm that all authors have read and approved the submission of the manuscript, the manuscript has not been published and is not being considered for publication elsewhere, in whole or in part, in any language, except as an abstract. We also declare no conflict of interest, no financial relationships with any industry (through investments, employment, consultancies, stock ownership, honoraria).

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