An immunohistochemical study of enteric nervous system in the chick model

Document Type : Research Articles


1 University of Tehran

2 Shahrekord University

3 Tehran University of Medical Sciences


The chick model is a useful research tool to investigate the development of the enteric nervous system (ENS). Recognition of appropriate markers for detection of chick enteric ganglia will allow better utilization of this model to study abnormalities of the ENS. This study aimed to validate a set of antibodies for avian ENS studies on wax sections. The specimens were taken from jejunum and colorectum of early post-hatching chicks, fixed in
4% buffered formaldehyde and stained using haematoxylin and eosin (H&E). Glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), synaptophysin and S-100 immunohistochemical biomarkers were employed on paraffin-embedded blocks to identify enteric ganglia. The immuno-reactivity scoring was recorded using a semi-quantitative fourtiered system (0, 1+, 2+, and 3+).
In jejunum specimens, the immune-reactivity of GFAP was significantly higher than both synaptophysin (p=0.001) and S-100 (p=0.001). There was also a significant difference
(p=0.03) between the immune-reactivity induced by NSE and S-100 in the jejunum samples.
Significant differences were observed between GFAP immuno-reactivity and both synaptophysin and S-100 (p=0.013; and p =0.005, respectively) in the samples collected from colorectum. The level of immuno-reactivity between NSE and both synaptophysin and S-100 biomarkers in the colorectal specimens were also different significantly (p=0.02 and 0.007,respectively). The results of the present work showed that GFAP and NSE biomarkers can be used with high immuno-reactivities to examine the chick enteric ganglia as an appropriate animal model in ENS developmental disorders.


Aisa, J., Lahoz, M.,Serrano, P. J.,Junquera,C.,Peg, M. T., and Vera-Gil, A. (1997)Intrinsic innervation of the chicken
lower digestive tract. Neurochemical Research 22, 1425-1435.
Aitken, R. N. C.(1958) A histochemical study of the stomach and intestine of the chicken. Journal of Anatomy 92, 453-466.
Amiel, J., Emison, E. S.,Barcelo, M. G.,Lantieri, F.,Burzynski, G.,Borrego,S.,Pelet, A.,Arnold, S.,Miao,X.,Griseri, P.,Brooks, A. S.,Antinolo,G.,dePontula, L.,Clement-Ziza,M.,Munnich, A.,Kashuk, C.,West,K.,Wong, K. K.,Lyonnet,S.,Chakravarti, A.,Tam, P.K.,Ceccherini, I.,Hofstra, R. M.,andFernandez, R. (2007) Hirschsprung disease, associated syndromes andgenetics: a review. Journal of Medical Genetics 45, 1-14.
Balaskas, C., and Gabella, G. (1997) Laminin immunoreactivity in enteric ganglia of the chick embryo. Cell and Tissue Research 289, 243-251.
Burns A.J. (2005) Migration of neural crestderived enteric nervous system precursor cells to and within the gastrointestinal tract. International Journal of Developmental Biology 49,143-150.
Dabbs, D. (2006) Diagnostic immunohistochemistry. Churchill Livingstone, China.
Doyle, A. M., Roberts, D. J., and Goldstein, A.M. (2004) Enteric nervous system patterning in the avian hindgut.Developmental Dynamics 229, 708-712.
Dzienis-Koronkiewicz, E., Debek, W. S., and Chyczewski, L. (2005) Use of synaptophysin immunohistochemistry in intestinal motility disorders. European Journal of Pediatric Surgery 15(6), 392-398.
Fenoglio-Preiser, C., Noffsinger, A.E.,Stemmermann, G. N.,Lantz, P. E.,and Isaacson, P. G. (2008) Gastrointestinal pathology: an atlas and text. Lippincott Williams & Wilkins,Baltimore.
Gershon, M. D., and Ratcliffe, E. M. (2004) Developmental biology of the enteric nervous system: pathogenesis of
Hirschsprung's disease and other congenital dysmotilities. Seminars in Pediatric Surgery 13, 224-235.
Goldstein, A. M., and Nagy, N. (2008)A bird's eye view of enteric nervous system development: lessons from the avian embryo. Pediatric Research 64, 329- 333.
Kapur, R. P.(2000) Developmental disorders of the enteric nervous system. Gut 47,81-83.
Kapur, R. P.(2006) Immunohistochemistry and the diagnosis of Hirschsprung disease.American Journal of Clinical Pathology 126, 9-12.
Karim, S., Hession, C., Marconi, S.,Gang, D.L., and Otis,C. N. (2006) The identification of ganglion cells in Hirschsprung disease by the immunohistochemical detection of ret oncoprotein. American Journal of Clinical Pathology 126, 49-54.
Kawana, T., Nada, K.,Ikeda, S.,Goto,S.,Hirose, R.,Taguchi, T.,Kubota,M.,Toyohara, T., and Shono, T. (1989) Distribution and localization of glial Fibrillary acidic protein in colons affected by Hirschsprung's disease. Journal of Pediatric Surgery 24, 448-
Li, J. C., Mi, K. H., Zhou, J. L., and Kuhnel,W. (2001) The development of colon innervation in trisomy 16 mice and
Hirschsprung's disease. World Journal of Gastroenterology 7, 16-21.
Maruccio, L., Lucini, C., Russo, F., Antonucci,R., and Castaldo, L. (2008)Thedevelopment of avian enteric nervous system: Distribution of arteminimmunoreactivity. Acta Histochemica 110, 163-171.
Memarzadeh, M., Talebi, A., Edalati, M.,Hosseinpour, M., and Vahidi, N. (2009) Hirschsprung's disease diagnosis: Comparison of immunohistochemical, haematoxylin and eosin staining. Journal of the Indian Association of Pediatric Surgeons 14, 59-62.
Metzger, M.(2010) Neurogenesis in the enteric nervous system.Archives Italiennes de Biologie 148, 73-83.
O'Donnell, A. N., and Puri, P. (2009) Hypoganglioniccolorectum in the chick embryo: a model of human hypoganglionosis. Pediatric Surgery International. 25, 885-888.
ParisiSalvi, E.,Vaccaro, R.,Baglaj, S. M., and Renda,T. (2004) Nervous system development in normal and atresic chick embryo intestine: an immunohistochemical study. Anatomy And Embryology 209,143-151.
Park, S. H., Min, H., Chi, J. G.,Park, K.W.,Yang, H. R., and Seo, J. K. (2005) Immunohistochemical studies of pediatric intestinal pseudo-obstruction:bcl2, a valuable biomarker to detect immature enteric ganglion cells.American Journal of Surgical Pathology 29, 1017-1024.
Porter, B. F., Storts, R. W.,Payne, H. R., and Edwards, J. F. (2007) Colonic ganglioneuromatosis in a horse.Veterinary Pathology 44, 207-210.
Robey, S. S., Kuhajda, F. P., and Yardley, J.H. (1988) Immunoperoxidase stains of ganglion cells and abnormal mucosal nerve proliferations in Hirschsprung disease. Human Pathology 19, 432- 437.
Rouleau, C., Matecki, S., Kalfa,N., Costes, V., and de Santa Barbara, P.(2009)Activation of MAP kinase (ERK1/2) in human neonatal colonic enteric nervous system. Neurogastroenterology and Motility 21, 207-214.
Ruiz, F. S., Alessl, A. C.,Chagas, C. A.,Pinto, G. A., ndVassallo,J. (2005) Immunohistochemistry in diagnostic veterinary pathology: a critical review Jornal Brasileiro de Patologia e Medicina Laboratorial 41, 263-270.
Ure, B. M., Holschneider, A. M., and Meier- Ruge, W. (1994) Neuronal intestinal malformations: a retro- and prospective study on 203 patients. European Journal of Pediatric Surgery 4, 279-286.
Wester, T., Olsson, Y., and Olsen, L. (1999) Expression of bcl-2 in enteric neurons in normal human bowel and Hirschsprung disease. Archives of Pathology & Laboratory Medicine 123, 1264-1268.
Yanlei Huang, Anupama, B., ShanZheng, Xianmin Xiao, and Lian Chen.(2011)The expression of enteric nerve markers and nerve innervation in total colonic aganglionosis. International Journal of Surgical Pathology 19, 303-308.