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What neurons hide behind calretinin immunoreactivity in the human gut?

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Abstract

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.

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References

  • Accili EA, Dhatt N, Buchan AM (1995) Neural somatostatin, vasoactive intestinal polypeptide and substance P in canine and human jejunum. Neurosci Lett 185:37–40

    Article  CAS  PubMed  Google Scholar 

  • Anlauf M, Schäfer MK, Eiden L, Weihe E (2003) Chemical coding of the human gastrointestinal nervous system: cholinergic, VIPergic, and catecholaminergic phenotypes. J Comp Neurol 459:90–111

    Article  CAS  PubMed  Google Scholar 

  • Barshack I, Fridman E, Goldberg I, Chowers Y, Kopolovic J (2004) The loss of calretinin expression indicates aganglionosis in Hirschsprung’s disease. J Clin Pathol 57:712–716

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Beck M, Schlabrakowski A, Schrödl F, Neuhuber W, Brehmer A (2009) ChAT and NOS in human myenteric neurons: co-existence and co-absence. Cell Tissue Res 338:37–51

    Article  CAS  PubMed  Google Scholar 

  • Ben-Horin S, Chowers Y (2008) Neuroimmunology of the gut: physiology, pathology, and pharmacology. Curr Opin Pharmacol 8:490–495

    Article  CAS  PubMed  Google Scholar 

  • Beyer J, Jabari S, Rau TT, Neuhuber W, Brehmer A (2013) Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons. Histochem Cell Biol 140:157–167

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A (2006) Structure of enteric neurons. Adv Anat Embryol Cell Biol 186:1–94

    CAS  PubMed  Google Scholar 

  • Brehmer A, Blaser B, Seitz G, Schrödl F, Neuhuber W (2004a) Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic, neurofilament immunoreactive myenteric neuron types of human small intestine. Histochem Cell Biol 121:13–20

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A, Croner R, Dimmler A, Papadopoulos T, Schrödl F, Neuhuber W (2004b) Immunohistochemical characterization of putative primary afferent (sensory) myenteric neurons in human small intestine. Auton Neurosci 112:49–59

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A, Lindig TM, Schrödl F, Neuhuber W, Ditterich D, Rexer M, Rupprecht H (2005) Morphology of enkephalin-immunoreactive myenteric neurons in the human gut. Histochem Cell Biol 123:131–138

    Article  CAS  PubMed  Google Scholar 

  • Brehmer A, Schrödl F, Neuhuber W (2006) Morphology of VIP/nNOS-immunoreactive myenteric neurons in the human gut. Histochem Cell Biol 125:557–565

    Article  CAS  PubMed  Google Scholar 

  • Brenneman DE (2007) Neuroprotection: a comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Peptides 28:1720–1726

    Article  CAS  PubMed  Google Scholar 

  • Costa M, Brookes SJ, Steele PA, Gibbins I, Burcher E, Kandiah CJ (1996) Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience 75:949–967

    Article  CAS  PubMed  Google Scholar 

  • Crowe R, Kamm MA, Burnstock G, Lennard-Jones JE (1992) Peptide-containing neurons in different regions of the submucous plexus of human sigmoid colon. Gastroenterology 102:461–467

    CAS  PubMed  Google Scholar 

  • Dhatt N, Buchan AM (1994) Colocalization of neuropeptides with calbindin DSek and NADPH diaphorase in the enteric nerve plexuses of normal human ileum. Gastroenterology 107:680–690

    Article  CAS  PubMed  Google Scholar 

  • Domoto T, Bishop AE, Oki M, Polak JM (1990) An in vitro study of the projections of enteric vasoactive intestinal polypeptide-immunoreactive neurons in the human colon. Gastroenterology 98:819–827

    Article  CAS  PubMed  Google Scholar 

  • Ekblad E, Bauer AJ (2004) Role of vasoactive intestinal peptide and inflammatory mediators in enteric neuronal plasticity. Neurogastroenterol Motil 16(Suppl 1):123–128

    Article  PubMed  Google Scholar 

  • Furness JB (2006) The enteric nervous system. Blackwell, Oxford

    Google Scholar 

  • Ganns D, Schrödl F, Neuhuber W, Brehmer A (2006) Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine. Histol Histopathol 21:41–51

    CAS  PubMed  Google Scholar 

  • Grider JR (1989) Identification of neurotransmitters regulating intestinal peristaltic reflex in humans. Gastroenterology 97:1414–1419

    CAS  PubMed  Google Scholar 

  • Guinard-Samuel V, Bonnard A, De Lagausie P, Philippe-Chomette P, Alberti C, El Ghoneimi A, Peuchmaur M, Berrebi-Binczak D (2009) Calretinin immunohistochemistry: a simple and efficient tool to diagnose Hirschsprung disease. Mod Pathol 22:1379–1384

    Article  CAS  PubMed  Google Scholar 

  • Holland SK, Ramalingam P, Podolsky RH, Reid-Nicholson MD, Lee JR (2011) Calretinin immunostaining as an adjunct in the diagnosis of Hirschsprung disease. Ann Diagn Pathol 15:323–328

    Article  PubMed  Google Scholar 

  • Jabari S, da Silveira AB, de Oliveira EC, Neto SG, Quint K, Neuhuber W, Brehmer A (2012a) Selective survival of calretinin- and vasoactive-intestinal-peptide-containing nerve elements in human chagasic submucosa and mucosa. Cell Tissue Res 349:473–481

    Article  CAS  PubMed  Google Scholar 

  • Jabari S, da Silveira AB, de Oliveira EC, Quint K, Neuhuber W, Brehmer A (2012b) Preponderance of inhibitory versus excitatory intramuscular nerve fibres in human chagasic megacolon. Int J Colorectal Dis 27:1181–1189

    Article  PubMed  Google Scholar 

  • Kapur RP, Reed RC, Finn LS, Patterson K, Johanson J, Rutledge JC (2009) Calretinin immunohistochemistry versus acetylcholinesterase histochemistry in the evaluation of suction rectal biopsies for Hirschsprung Disease. Pediatr Dev Pathol 12:6–15

    Article  PubMed  Google Scholar 

  • Knowles CH, De Giorgio R, Kapur RP, Bruder E, Farrugia G, Geboes K, Gershon MD, Hutson J, Lindberg G, Martin JE, Meier-Ruge WA, Milla PJ, Smith VV, Vandervinden JM, Veress B, Wedel T (2009) Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group. Acta Neuropathol 118:271–301

    Article  PubMed  Google Scholar 

  • Kramer K, da Silveira AB, Jabari S, Kressel M, Raab M, Brehmer A (2011) Quantitative evaluation of neurons in the mucosal plexus of adult human intestines. Histochem Cell Biol 136:1–9

    Article  CAS  PubMed  Google Scholar 

  • Krammer HJ, Zhang M, Kuhnel W (1994) Distribution of NADPH-diaphorase-positive neurons in the enteric nervous system of the human colon. Ann Anat 176:137–141

    Article  CAS  PubMed  Google Scholar 

  • Kustermann A, Neuhuber W, Brehmer A (2011) Calretinin and somatostatin immunoreactivities label different human submucosal neuron populations. Anat Rec (Hoboken) 294:858–869

    Article  CAS  Google Scholar 

  • Lindig TM, Kumar V, Kikinis R, Pieper S, Schrödl F, Neuhuber WL, Brehmer A (2009) Spiny versus stubby: 3D reconstruction of human myenteric (type I) neurons. Histochem Cell Biol 131:1–12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mayer B, John M, Bohme E (1990) Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum: cofactor-role of tetrahydrobiopterin. FEBS Lett 277:215–219

    Article  CAS  PubMed  Google Scholar 

  • Mayer B, John M, Heinzel B, Werner ER, Wachter H, Schultz G, Bohme E (1991) Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase. FEBS Lett 288:187–191

    Article  CAS  PubMed  Google Scholar 

  • Morris MI, Soglio DB, Ouimet A, Aspirot A, Patey N (2013) A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis. J Pediatr Surg 48:1037–1043

    Article  PubMed  Google Scholar 

  • Neunlist M, Toumi F, Oreschkova T, Denis M, Leborgne J, Laboisse CL, Galmiche JP, Jarry A (2003) Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways. Am J Physiol Gastrointest Liver Physiol 285:G1028–G1036

    CAS  PubMed  Google Scholar 

  • Neunlist M, Van Landeghem L, Mahe MM, Derkinderen P, des Varannes SB, Rolli-Derkinderen M (2013) The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease. Nat Rev Gastroenterol Hepatol 10:90–100

    Article  CAS  PubMed  Google Scholar 

  • Porter AJ, Wattchow DA, Brookes SJ, Costa M (1999) Projections of nitric oxide synthase and vasoactive intestinal polypeptide-reactive submucosal neurons in the human colon. J Gastroenterol Hepatol 14:1180–1187

    Article  CAS  PubMed  Google Scholar 

  • Schnell SA, Staines WA, Wessendorf MW (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47:719–730

    Article  CAS  PubMed  Google Scholar 

  • Schuy J, Schlabrakowski A, Neuhuber W, Brehmer A (2011) Quantitative estimation and chemical coding of spiny type I neurons in human intestines. Cells Tissues Organs 193:195–206

    Article  CAS  PubMed  Google Scholar 

  • Timmermans JP, Barbiers M, Scheuermann DW, Bogers JJ, Adriaensen D, Fekete E, Mayer B, Van Marck EA, De Groodt-Lasseel MH (1994) Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract. Cell Tissue Res 275:235–245

    Article  CAS  PubMed  Google Scholar 

  • Toumi F, Neunlist M, Cassagnau E, Parois S, Laboisse CL, Galmiche JP, Jarry A (2003) Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co-culture model. Neurogastroenterol Motil 15:239–242

    Article  CAS  PubMed  Google Scholar 

  • Walters JR, Bishop AE, Facer P, Lawson EM, Rogers JH, Polak JM (1993) Calretinin and calbindin-D28K immunoreactivity in the human gastrointestinal tract. Gastroenterology 104:1381–1389

    CAS  PubMed  Google Scholar 

  • Weidmann S, Schrödl F, Neuhuber W, Brehmer A (2007) Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine. Histochem Cell Biol 128:399–407

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The excellent technical assistance of Karin Löschner, Stefanie Link, Anita Hecht, Andrea Hilpert, and Hedwig Symowski is gratefully acknowledged. Furthermore, we thank Jochen Lennerz (Ulm) for fruitful discussion.

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Authors and Affiliations

  1. Institute of Anatomy I, University of Erlangen-Nuremberg, Krankenhausstraße 9, 91054, Erlangen, Germany

    Nicholas Beuscher, Samir Jabari, Winfried Neuhuber & Axel Brehmer

  2. Institute of Pathology, University of Erlangen-Nuremberg, Krankenhausstraße 8-10, 91054, Erlangen, Germany

    Johanna Strehl

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  1. Nicholas Beuscher
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  2. Samir Jabari
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Correspondence to Axel Brehmer.

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Beuscher, N., Jabari, S., Strehl, J. et al. What neurons hide behind calretinin immunoreactivity in the human gut?. Histochem Cell Biol 141, 393–405 (2014). https://doi.org/10.1007/s00418-013-1163-0

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